Immunodeficiency recombinant poxvirus

ABSTRACT

Attenuated recombinant viruses containing DNA encoding an immunodeficiency virus and/or CTL antigen, as well as methods and compositions employing the viruses, expression products therefrom, and antibodies generated from the viruses or expression products, are disclosed and claimed. The recombinant viruses can be NYVAC or ALVAC recombinant viruses. The DNA can code for at least one of: HIV1gag(+pro) (IIIB), gp120(MN) (+transmembrane), nef (BRU)CTL, pol(IIIB)CTL, ELDKWA or LDKW epitopes, preferably HIV1gag(+pro) (IIIB), gp120(MN) (+transmembrane), two (2) nef(BRU)CTL and three (3) pol(IIIB)CTL epitopes; or two ELDKWA in gp120 V3 or another region or in gp160. The two (2) nef(BRU)CTL and three (3) pol(IIIB)CTL epitopes are preferably CTL1, CTL2, pol1, pol2 and pol3. The recombinant viruses and gene products therefrom and antibodies generated by the viruses and gene products have several preventive, therapeutic and diagnostic uses. DNA from the recombinant viruses are useful as probes or, for generating PCR primers or for immunization. Also disclosed and claimed are HIV immunogens and modified gp160 and gp120.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/441,788,filed May 20, 2003, which in turn is a divisional of application Ser.No. 09/136,159, filed Aug. 14, 1998, now U.S. Pat. No. 6,596,279, whichin turn is a divisional of application Ser. No. 08/417,210, filed Apr.5, 1995, now U.S. Pat. No. 5,863,542, which in turn is acontinuation-in-part of application Ser. No. 08/223,842, filed Apr. 6,1994, abandoned, which in turn is a continuation-in-part of applicationSer. No. 07/897,382, filed Jun. 11, 1992, abandoned, which in turn is acontinuation-in-part of application Ser. No. 07/715,921, filed Jun. 14,1991, abandoned. This application is also a continuation-in-part ofapplication Ser. No. 08/105,483, filed Aug. 12, 1993, now U.S. Pat. No.5,494,807 which in turn is a continuation of application Ser. No.07/847,951, filed Mar. 6, 1992, abandoned, which in turn is acontinuation-in-part of application Ser. No. 07/713,967, filed Jun. 11,1991, abandoned, which in turn is a continuation in part of applicationSer. No. 07/666,056, filed Mar. 7, 1991, abandoned. Mention is also madeof co-pending application Ser. No. 08/184,009, filed Jan. 19, 1994, nowU.S. Pat. No. 5,833,975, as a continuation-in-part of application Ser.No. 08/007,115, filed Jan. 21, 1993, abandoned. Each of theaforementioned and above-referenced applications is hereby incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to a modified poxvirus and to methods ofmaking and using the same. More in particular, the invention relates toimproved vectors for the insertion and expression of foreign genes foruse as safe immunization vehicles to elicit an immune response againstimmunodeficiency virus. Thus, the invention relates to a recombinantpoxvirus, which virus expresses gene products of immunodeficiency virusand to immunogenic compositions which induce an immunological responseagainst immunodeficiency virus infections when administered to a host,or in vitro (e.g. ex vivo modalities) as well as to the products ofexpression of the poxvirus which by themselves are useful for elicitingan immune response e.g., raising antibodies, which antibodies are usefulagainst immunodeficiency virus infection, in either seropositive orseronegative individuals, or are useful if isolated from an animal orhuman for preparing a diagnostic kit, test or assay for the detection ofthe virus or infected cells.

Several publications are referenced in this application. Full citationto these references is found at the end of the specification immediatelypreceding the claims or where the publication is mentioned; and each ofthese publications is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Vaccinia virus and more recently other poxviruses have been used for theinsertion and expression of foreign genes. The basic technique ofinserting foreign genes into live infectious poxvirus involvesrecombination between pox DNA sequences flanking a foreign geneticelement in a donor plasmid and homologous sequences present in therescuing poxvirus (Piccini et al., 1987).

Specifically, the recombinant poxviruses are constructed in two stepsknown in the art and analogous to the methods for creating syntheticrecombinants of poxviruses such as the vaccinia virus and avipox virusdescribed in U.S. Pat. Nos. 4,769,330, 4,772,848, 4,603,112, 5,100,587,and 5,179,993, the disclosures of which are incorporated herein byreference.

First, the DNA gene sequence to be inserted into the virus, particularlyan open reading frame from a non-pox source, is placed into an E. coliplasmid construct into which DNA homologous to a section of DNA of thepoxvirus has been inserted. Separately, the DNA gene sequence to beinserted is ligated to a promoter. The promoter-gene linkage ispositioned in the plasmid construct so that the promoter-gene linkage isflanked on both ends by DNA homologous to a DNA sequence flanking aregion of pox DNA containing a nonessential locus. The resulting plasmidconstruct is then amplified by growth within E. coli bacteria (Clewell,1972) and isolated (Clewell et al., 1969; Maniatis et al., 1982).

Second, the isolated plasmid containing the DNA gene sequence to beinserted is transfected into a cell culture, e.g. chick embryofibroblasts, along with the poxvirus. Recombination between homologouspox DNA in the plasmid and the viral genome respectively gives apoxvirus modified by the presence, in a nonessential region of itsgenome, of foreign DNA sequences. The term “foreign” DNA designatesexogenous DNA, particularly DNA from a non-pox source, that codes forgene products not ordinarily produced by the genome into which theexogenous DNA is placed.

Genetic recombination is in general the exchange of homologous sectionsof DNA between two strands of DNA. In certain viruses RNA may replaceDNA. Homologous sections of nucleic acid are sections of nucleic acid(DNA or RNA) which have the same sequence of nucleotide bases.

Genetic recombination may take place naturally during the replication ormanufacture of new viral genomes within the infected host cell. Thus,genetic recombination between viral genes may occur during the viralreplication cycle that takes place in a host cell which is co-infectedwith two or more different viruses or other genetic constructs. Asection of DNA from a first genome is used interchangeably inconstructing the section of the genome of a second co-infecting virus inwhich the DNA is homologous with that of the first viral genome.

However, recombination can also take place between sections of DNA indifferent genomes that are not perfectly homologous. If one such sectionis from a first genome homologous with a section of another genomeexcept for the presence within the first section of, for example, agenetic marker or a gene coding for an antigenic determinant insertedinto a portion of the homologous DNA, recombination can still take placeand the products of that recombination are then detectable by thepresence of that genetic marker or gene in the recombinant viral genome.Additional strategies have recently been reported for generatingrecombinant vaccinia virus.

Successful expression of the inserted DNA genetic sequence by themodified infectious virus requires two conditions. First, the insertionmust be into a nonessential region of the virus in order that themodified virus remain viable. The second condition for expression ofinserted DNA is the presence of a promoter in the proper relationship tothe inserted DNA. The promoter must be placed so that it is locatedupstream from the DNA sequence to be expressed.

Vaccinia virus has been used successfully to immunize against smallpox,culminating in the worldwide eradication of smallpox in 1980. In thecourse of its history, many strains of vaccinia have arisen. Thesedifferent strains demonstrate varying immunogenicity and are implicatedto varying degrees with potential complications, the most serious ofwhich are post-vaccinial encephalitis and generalized vaccinia(Behbehani, 1983).

With the eradication of smallpox, a new role for vaccinia becameimportant, that of a genetically engineered vector for the expression offoreign genes. Genes encoding a vast number of heterologous antigenshave been expressed in vaccinia, often resulting in protective immunityagainst challenge by the corresponding pathogen (reviewed in Tartagliaet al., 1990a).

The genetic background of the vaccinia vector has been shown to affectthe protective efficacy of the expressed foreign immunogen. For example,expression of Epstein Barr Virus (EBV) gp340 in the Wyeth vaccine strainof vaccinia virus did not protect cottontop tamarins against EBV virusinduced lymphoma, while expression of the same gene in the WR laboratorystrain of vaccinia virus was protective (Morgan et al., 1988).

A fine balance between the efficacy and the safety of a vacciniavirus-based recombinant vaccine candidate is extremely important. Therecombinant virus must present the immunogen(s) in a manner that elicitsa protective immune response in the vaccinated animal but lacks anysignificant pathogenic properties. Therefore attenuation of the vectorstrain would be a highly desirable advance over the current state oftechnology.

A number of vaccinia genes have been identified which are non-essentialfor growth of the virus in tissue culture and whose deletion orinactivation reduces virulence in a variety of animal systems.

The gene encoding the vaccinia virus thymidine kinase (TK) has beenmapped (Hruby et al., 1982) and sequenced (Hruby et al., 1983; Weir etal., 1983). Inactivation or complete deletion of the thymidine kinasegene does not prevent growth of vaccinia virus in a wide variety ofcells in tissue culture. TK⁻ vaccinia virus is also capable ofreplication in vivo at the site of inoculation in a variety of hosts bya variety of routes.

It has been shown for herpes simplex virus type 2 that intravaginalinoculation of guinea pigs with TK⁻ virus resulted in significantlylower virus titers in the spinal cord than did inoculation with TK⁺virus (Stanberry et al., 1985). It has been demonstrated thatherpesvirus encoded TK activity in vitro was not important for virusgrowth in actively metabolizing cells, but was required for virus growthin quiescent cells (Jamieson et al., 1974).

Attenuation of TK⁻ vaccinia has been shown in mice inoculated by theintracerebral and intraperitoneal routes (Buller et al., 1985).Attenuation was observed both for the WR neurovirulent laboratory strainand for the Wyeth vaccine strain. In mice inoculated by the intradermalroute, TK⁻ recombinant vaccinia generated equivalent anti-vaccinianeutralizing antibodies as compared with the parental TK⁺ vacciniavirus, indicating that in this test system the loss of TK function doesnot significantly decrease immunogenicity of the vaccinia virus vector.Following intranasal inoculation of mice with TK⁻ and TK⁺ recombinantvaccinia virus (WR strain), significantly less dissemination of virus toother locations, including the brain, has been found (Taylor et al.,1991a).

Another enzyme involved with nucleotide metabolism is ribonucleotidereductase. Loss of virally encoded ribonucleotide reductase activity inherpes simplex virus (HSV) by deletion of the gene encoding the largesubunit was shown to have no effect on viral growth and DNA synthesis individing cells in vitro, but severely compromised the ability of thevirus to grow on serum starved cells (Goldstein et al., 1988). Using amouse model for acute HSV infection of the eye and reactivatable latentinfection in the trigeminal ganglia, reduced virulence was demonstratedfor HSV deleted of the large subunit of ribonucleotide reductase,compared to the virulence exhibited by wild type HSV (Jacobson et al.,1989).

Both the small (Slabaugh et al., 1988) and large (Schmidtt et al., 1988)subunits of ribonucleotide reductase have been identified in vacciniavirus. Insertional inactivation of the large subunit of ribonucleotidereductase in the WR strain of vaccinia virus leads to attenuation of thevirus as measured by intracranial inoculation of mice (Child et al.,1990).

The vaccinia virus hemagglutinin gene (HA) has been mapped and sequenced(Shida, 1986). The HA gene of vaccinia virus is nonessential for growthin tissue culture (Ichihashi et al., 1971). Inactivation of the HA geneof vaccinia virus results in reduced neurovirulence in rabbitsinoculated by the intracranial route and smaller lesions in rabbits atthe site of intradermal inoculation (Shida et al., 1988). The HA locuswas used for the insertion of foreign genes in the WR strain (Shida etal., 1987), derivatives of the Lister strain (Shida et al., 1988) andthe Copenhagen strain (Guo et al., 1989) of vaccinia virus. RecombinantHA vaccinia virus expressing foreign genes have been shown to beimmunogenic (Guo et al., 1989; Itamura et al., 1990; Shida et al., 1988;Shida et al., 1987) and protective against challenge by the relevantpathogen (Guo et al., 1989; Shida et al., 1987).

Cowpox virus (Brighton red strain) produces red (hemorrhagic) pocks onthe chorioallantoic membrane of chicken eggs. Spontaneous deletionswithin the cowpox genome generate mutants which produce white pocks(Pickup et al., 1984). The hemorrhagic function (u) maps to a 38 kDaprotein encoded by an early gene (Pickup et al., 1986). This gene, whichhas homology to serine protease inhibitors, has been shown to inhibitthe host inflammatory response to cowpox virus (Palumbo et al., 1989)and is an inhibitor of blood coagulation.

The u gene is present in WR strain of vaccinia virus (Kotwal et al.,1989b). Mice inoculated with a WR vaccinia virus recombinant in whichthe u region has been inactivated by insertion of a foreign gene producehigher antibody levels to the foreign gene product compared to miceinoculated with a similar recombinant vaccinia virus in which the u geneis intact (Zhou et al., 1990). The u region is present in a defectivenonfunctional form in Copenhagen strain of vaccinia virus (open readingframes B13 and B14 by the terminology reported in Goebel et al.,1990a,b).

Cowpox virus is localized in infected cells in cytoplasmic A typeinclusion bodies (ATI) (Kato et al., 1959). The function of ATI isthought to be the protection of cowpox virus virions duringdissemination from animal to animal (Bergoin et al., 1971). The ATIregion of the cowpox genome encodes a 160 kDa protein which forms thematrix of the ATI bodies (Funahashi et al., 1988; Patel et al., 1987).Vaccinia virus, though containing a homologous region in its genome,generally does not produce ATI. In WR strain of vaccinia, the ATI regionof the genome is translated as a 94 kDa protein (Patel et al., 1988). InCopenhagen strain of vaccinia virus, most of the DNA sequencescorresponding to the ATI region are deleted, with the remaining 3′ endof the region fused with sequences upstream from the ATI region to formopen reading frame (ORF) A26L (Goebel et al., 1990a,b).

A variety of spontaneous (Altenburger et al., 1989; Drillien et al.,1981; Lai et al., 1989; Moss et al., 1981; Paez et al., 1985; Panicaliet al., 1981) and engineered (Perkus et al., 1991; Perkus et al., 1989;Perkus et al., 1986) deletions have been reported near the left end ofthe vaccinia virus genome. A WR strain of vaccinia virus with a 10 kbspontaneous deletion (Moss et al., 1981; Panicali et al., 1981) wasshown to be attenuated by intracranial inoculation in mice (Buller etal., 1985). This deletion was later shown to include 17 potential ORFs(Kotwal et al., 1988b). Specific genes within the deleted region includethe virokine N1L and a 35 kDa protein (C3L, by the terminology reportedin Goebel et al., 1990a,b). Insertional inactivation of N1L reducesvirulence by intracranial inoculation for both normal and nude mice(Kotwal et al., 1989a). The 35 kDa protein is secreted like N1L into themedium of vaccinia virus infected cells. The protein contains homologyto the family of complement control proteins, particularly thecomplement 4B binding protein (C4 bp) (Kotwal et al., 1988a). Like thecellular C4 bp, the vaccinia 35 kDa protein binds the fourth componentof complement and inhibits the classical complement cascade (Kotwal etal., 1990). Thus the vaccinia 35 kDa protein appears to be involved inaiding the virus in evading host defense mechanisms.

The left end of the vaccinia genome includes two genes which have beenidentified as host range genes, K1L (Gillard et al., 1986) and C7L(Perkus et al., 1990). Deletion of both of these genes reduces theability of vaccinia virus to grow on a variety of human cell lines(Perkus et al., 1990).

Two additional vaccine vector systems involve the use of naturallyhost-restricted poxviruses, avipoxviruses. Both fowlpoxvirus (FPV) andcanarypoxvirus (CPV) have been engineered to express foreign geneproducts. Fowlpox virus (FPV) is the prototypic virus of the Avipoxgenus of the Poxvirus family. The virus causes an economically importantdisease of poultry which has been well controlled since the 1920's bythe use of live attenuated vaccines. Replication of the avipox virusesis limited to avian species (Matthews, 1982) and there are no reports inthe literature of avipoxvirus causing a productive infection in anynon-avian species including man. This host restriction provides aninherent safety barrier to transmission of the virus to other speciesand makes use of avipoxvirus based vaccine vectors in veterinary andhuman applications an attractive proposition.

FPV has been used advantageously as a vector expressing antigens frompoultry pathogens. The hemagglutinin protein of a virulent avianinfluenza virus was expressed in an FPV recombinant (Taylor et al.,1988a). After inoculation of the recombinant into chickens and turkeys,an immune response was induced which was protective against either ahomologous or a heterologous virulent influenza virus challenge (Tayloret al., 1988a). FPV recombinants expressing the surface glycoproteins ofNewcastle Disease Virus have also been developed (Taylor et al., 1990;Edbauer et al., 1990).

Despite the host-restriction for replication of FPV and CPV to aviansystems, recombinants derived from these viruses were found to expressextrinsic proteins in cells of nonavian origin. Further, suchrecombinant viruses were shown to elicit immunological responsesdirected towards the foreign gene product and where appropriate wereshown to afford protection from challenge against the correspondingpathogen (Tartaglia et al., 1993a,b; Taylor et al., 1992; 1991b; 1988b).

In 1983, human immunodeficiency virus type 1 (HIV1) was identified asthe causative agent of AIDS. Twelve years later, despite a massive,worldwide effort, an effective HIV1 vaccine is still not available.Recently, however, several reports have suggested that an efficaciousHIV1 vaccine may be attainable. For example, macaques have beenprotected against a simian immunodeficiency virus (SIV) challenge by avaccination protocol involving a primary immunization with a vacciniavirus recombinant expressing the SIV gp160 glycoprotein and a boosterimmunization with purified SIV gp160 glycoprotein (Hu et al., 1992). Inaddition, chimpanzees have been protected against an HIV1 challenge withan HIV1 gp120 subunit vaccine (Berman et al, 1990). Chimps have alsobeen protected against an HIV1 challenge by a vaccination protocolinvolving multiple injections of either inactivated HIV1, gp160 and/orV3 peptide or gp160, p17 (a Gag protein) and/or V3 peptide (Girard etal., 1991). A similar protocol involving multiple injections of gp160,p17, p24 (a Gag protein), Vif, Nef and/or V3 peptide has also protectedchimps against a challenge of HIV1-infected cells (Fultz et al., 1992).Furthermore, chimps have been passively protected by the infusion ofHIV1 V3-specific antibodies (Emini et al., 1992).

Most of these vaccination protocols have focused on eliciting an immuneresponse against the HIV1 or SIV envelope glycoprotein, or morespecifically, against the V3 epitope of the envelope glycoprotein.Unfortunately, different strains of HIV1 exhibit extensive genetic andantigenic variability, especially in the envelope glycoprotein.Therefore, an effective HIV1 vaccine may need to elicit an immuneresponse against more than one HIV1 antigen, or one epitope of one HIV1antigen.

Contrary to the extensive sequence variability observed in B-cellepitopes, T-cell epitopes are relatively conserved. For example,cytotoxic T-lymphocytes (CTL) clones, isolated from an HIV1-seronegativeindividual vaccinated with a vaccinia virus recombinant expressing HIV1gp160 (LAI strain) and boosted with purified HIV1 gp160 (LAI), lysetarget cells expressing the HIV1 MN or RF envelope glycoprotein asefficiently as cells expressing the HIV1 LAI envelope glycoprotein(Hammond et al., 1992). Therefore, a vaccine that elicits an immuneresponse against relatively conserved T-cell epitopes may not only bemore efficacious against a homologous challenge, but also moreefficacious against a heterologous challenge.

HIV1-seronegative individuals have been vaccinated with an ALVACrecombinant (vCP125) expressing HIV1 gp160, in a prime-boost protocolsimilar to the regimen used to vaccinate macaques against SIV. TheseALVAC-based protocols demonstrated the ability of vCP125 to elicit HIV1envelope-specific CD8⁺ CTLs and to enhance envelope-specific humoralresponses observed following a subunit booster (Pialoux et al., 1995).These results justify the rationale for a recombinant ALVAC-based HIV1vaccine.

Individuals infected with human immunodeficiency virus type 1 (HIV1)initially generate a relatively dynamic and extensive antiviral immuneresponse, including HIV1-specific neutralizing antibodies andHIV1-specific CTLs. Despite these responses, however, the vast majorityof HIV1-infected people eventually succumb to HIV1-associated diseases.Since the immune response generated by most HIV1-infected people is notprotective, generation of an effective immune response may necessitatethat the immune response be modulated or redirected against HIV1epitopes that are not normally or efficiently seen by HIV1-infectedindividuals.

Approximately 40% of the HIV1-specific antibody in HIV1-seropositiveindividuals capable of binding HIV1-infected cells is specific to thethird variable region (V3) of the HIV1 envelope glycoprotein (Spear etal, 1994). These results indicate that the V3 loop is 1) highlyimmunogenic and 2) exposed on the surface of infected cells. The aminoacid sequence of the V3 loop varies considerably between different HIV1isolates. Therefore, a moderate level of sequence variation does notappear to alter the structure or immunogenicity of this region of theenvelope glycoprotein. Since the V3 loop is highly immunogenic and itsstructure and immunogenicity is not severely affected by sequencevariation, this region of the envelope glycoprotein may be useful as animmunogenic platform for presenting normally non-immunogenic linear HIV1epitopes or heterologous epitopes to the immune system.

Sera from HIV1-seropositive individuals can neutralize lab-adaptedstrains of HIV1. These sera can also neutralize primary HIV1 isolates(although 100× higher titers are required). Conversely, sera fromindividuals vaccinated with HIV1 gp120 can neutralize lab-adaptedstrains of HIV1 (although 10× higher titers relative to sera fromseropositive individuals are required), but can not neutralize (atassayable levels) primary isolates (Hanson, 1994). A significant portionof the neutralizing activity found in sera from seropositive andgp120-vaccinated individuals appears to be specific to the V3 loop(Spear et al., 1994; Berman et al., 1994). Since the V3 loop ishypervariable and since antibodies against this region may notneutralize primary isolates or heterologous strains of HIV1, it may benecessary to develop vaccines that elicit an immune response againstepitopes other than the V3 loop, epitopes that can neutralize a broadspectrum of HIV1 strains, including primary isolates.

A monoclonal antibody capable of neutralizing primary HIV1 isolates, aswell as a broad spectrum of lab-adapted HIV1 strains, has been isolated(Conley et al., 1994; Katinger et al., 1992). The epitope recognized bythis monoclonal antibody has been mapped between amino acids 662 and 667of HIV1 gp41 and has the amino acid sequence, ELDKWA (Buchacher et al,1994). Approximately 80% of the HIV1 strains from which sequenceinformation has been derived, including strains from the various HIV1clades, express the core binding sequence of this epitope, LDKW (Conleyet al., 1994). Therefore, unlike the V3 loop, this epitope appears to berelatively well conserved. Unfortunately, this epitope does not appearto be very immunogenic in its normal configuration. Only approximately50% of HIV1-seropositive individuals have a detectable antibody responseto the region of gp41 containing this epitope (Broliden et al., 1992).

It can thus be appreciated that provision of an immunodeficiency virusrecombinant poxvirus, and of an immunogenic composition which induces animmunological response against immunodeficiency virus infections whenadministered to host, particularly a composition having enhanced safetysuch as NYVAC or ALVAC based recombinants containing coding for any orall of HIV1gag(+pro) (IIIB), gp120(MN) (+transmembrane), nef(BRU)CTLepitopes, pol(IIIB)CTL epitopes; for instance, HIV1gag(+pro) (IIIB),gp120(MN) (+transmembrane), nefCTL1, nefCTL2, pol1(PolCTL1),pol2(PolCTL2), pol3(PolCTL3), ELDKWA or LDKW epitopes (SEQ ID NOS: 147and 148), especially in an immunogenic configuration, or any combinationthereof, for example all of them in combination, would be a highlydesirable advance over the current state of technology.

OBJECTS AND SUMMARY OF THE INVENTION

It is therefore an object of this invention to provide modifiedrecombinant viruses, which viruses have enhanced safety, and to providea method of making such recombinant viruses.

It is an additional object of this invention to provide a recombinantpoxvirus antigenic, vaccine or immunological composition having anincreased level of safety compared to known recombinant poxvirusvaccines.

It is a further object of this invention to provide a modified vectorfor expressing a gene product in a host, wherein the vector is modifiedso that it has attenuated virulence in the host.

It is another object of this invention to provide a method forexpressing a gene product in a cell cultured in vitro using a modifiedrecombinant virus or modified vector having an increased level ofsafety.

These and other objects and advantages of the present invention willbecome more readily apparent after consideration of the following.

In one aspect, the present invention relates to a modified recombinantvirus having inactivated virus-encoded genetic functions so that therecombinant virus has attenuated virulence and enhanced safety. Thefunctions can be non-essential, or associated with virulence. The virusis advantageously a poxvirus, particularly a vaccinia virus or an avipoxvirus, such as fowlpox virus and canarypox virus. The modifiedrecombinant virus can include, within a non-essential region of thevirus genome, a heterologous DNA sequence which encodes an antigen orepitope derived from immunodeficiency virus and/or CTL epitope such as,e.g., HIV1gag(+pro) (IIIB), gp120(MN) (+transmembrane), nef(BRU)CTL,pol(IIIB)CTL, ELDKWA, LDKW epitopes or any combination thereof,preferably all of them in combination.

In another aspect, the present invention relates to an antigenic,immunological or vaccine composition or a therapeutic composition forinducing an antigenic or immunological response in a host animalinoculated with the composition, said vaccine including a carrier and amodified recombinant virus having inactivated nonessential virus-encodedgenetic functions so that the recombinant virus has attenuated virulenceand enhanced safety. The virus used in the composition according to thepresent invention is advantageously a poxvirus, particularly a vacciniavirus or an avipox virus, such as fowlpox virus and canarypox virus. Themodified recombinant virus can include, within a non-essential region ofthe virus genome, a heterologous DNA sequence which encodes an antigenicprotein, e.g., derived from immunodeficiency virus and/or CTL such as,HIV1gag(+pro) (IIIB), gp120 (MN) (+transmembrane), nef(BRU)CTL,pol(IIIB)CTL, ELDKWA, LDKW epitopes or any combination thereof,preferably all of them in combination.

In yet another aspect, the present invention relates to an immunogeniccomposition containing a modified recombinant virus having inactivatednonessential virus-encoded genetic functions so that the recombinantvirus has attenuated virulence and enhanced safety. The modifiedrecombinant virus includes, within a non-essential region of the virusgenome, a heterologous DNA sequence which encodes an antigenic protein(e.g., derived from an immunodeficiency virus and/or CTL such as,HIV1gag(+pro) (IIIB), gp120(MN) (+transmembrane), nef(BRU)CTL,pol(IIIB)CTL, ELDKWA, LDKW epitopes or any combination thereof,preferably all of them in combination) wherein the composition, whenadministered to a host, is capable of inducing an immunological responsespecific to the antigen.

In a further aspect, the present invention relates to a method forexpressing a gene product in a cell (e.g. peripheral blood mononuclearcells (PBMCs) or lymph node mononuclear cells (LNMC) in vitro byintroducing into the cell a modified recombinant virus having attenuatedvirulence and coenhanced safety. The modified recombinant virus caninclude, within a nonessential region of the virus genome, aheterologous DNA sequence which encodes an antigenic protein, e.g.derived from an immunodeficiency virus such as HIV/gag (+pro) (IIIB),gp120(MN) (+transmembrane), nef (BRU)CTL, pol (IIIB)CTL, ELDKWA, LDKWepitopes or any combination thereof, preferably all of them incombination. The cells can then be reinfused directly into theindividual or used to amplify specific CD8⁺ CTL reactivities forreinfusion (Ex vivo therapy).

In a further aspect, the present invention relates to a method forexpressing a gene product in a cell cultured in vitro by introducinginto the cell a modified recombinant virus having attenuated virulenceand enhanced safety. The modified recombinant virus can include, withina non-essential region of the virus genome, a heterologous DNA sequencewhich encodes an antigenic protein, e.g., derived from aimmunodeficiency virus such as HIV1gag (+pro) (IIIB), gp120(MN)(+transmembrane), nef(BRU)CTL, pol(IIIB)CTL, ELDKWA, LDKW epitopes orany combination thereof, preferably all of them in combination. Theproduct can then be administered to individuals or animals to stimulatean immune response. The antibodies raised can be useful in individualsfor the prevention or treatment of immunodeficiency virus and, theantibodies from animals can be used in diagnostic kits, assays or teststo determine the presence or absence in a sample such as sera ofimmunodeficiency virus or CTL antigens (and therefore the absence orpresence of the virus of an immune response to the virus or antibodies).

In a still further aspect, the present invention relates to a modifiedrecombinant virus having nonessential virus-encoded genetic functionsinactivated therein so that the virus has attenuated virulence, andwherein the modified recombinant virus further contains DNA from aheterologous source in a nonessential region of the virus genome. TheDNA can code for an immunodeficiency virus and/or CTL antigen such asHIV1gag(+pro) (IIIB), gp120(MN) (+transmembrane), nef(BRU)CTL,pol(IIIB)CTL, ELDKWA, LDKW epitopes or any combination thereof,preferably all of them in combination. In particular, the geneticfunctions are inactivated by deleting an open reading frame encoding avirulence factor or by utilizing naturally host restricted viruses. Thevirus used according to the present invention is advantageously apoxvirus, particularly a vaccinia virus or an avipox virus, such asfowlpox virus and canarypox virus. Advantageously, the open readingframe is selected from the group consisting of J2R, B13R+B14R, A26L,A56R, C7L-K1L, and I4L (by the terminology reported in Goebel et al.,1990a,b); and, the combination thereof. In this respect, the openreading frame comprises a thymidine kinase gene, a hemorrhagic region,an A type inclusion body region, a hemagglutinin gene, a host range generegion or a large subunit, ribonucleotide reductase; or, the combinationthereof. The modified Copenhagen strain of vaccinia virus is identifiedas NYVAC (Tartaglia et al., 1992). However, the COPAK strain can also beused in the practice of the invention.

Most preferably, in recombinant viruses of the invention, the exogenousDNA codes for HIV1gag(+pro) (IIIB), gp120(MN) (+transmembrane), two (2)nef(BRU)CTL and three (3) pol(IIIB)CTL epitopes; or, the exogenous DNAcodes for the ELDKWA or LDKW epitopes, and, is inserted so as to beexpressed in a region of gp120 or gp160 (i.e., the exogenous DNA codesfor a ELDKWA or LDKW modified gp120 or gp160, for instance ELDKWA orLDKW or repeats of either or both in the V3 loop) such that the epitopeis expressed in an immunogenic configuration. In this most preferredembodiment it is even more preferred that the two (2) nef(BRU)CTL andthree (3) pol(IIIB)CTL epitopes are CTL1, CTL2, poll, pol2, and pol3. Inanother most preferred embodiment the exogenous DNA codes for HIV1gp120+TM in which the V3 loop has been modified to contain at least one,and preferably two ELDKWA epitopes.

In further embodiments, the invention comprehends HIV immunogens andmodified gp160 or gp120. Thus, the invention includes an HIV immunogenpreferably selected from the group consisting of: HIV1gag(+pro) (IIIB),gp120(MN) (+transmembrane), nef (BRU)CTL, pol(IIIB)CTL, and ELDKWA orLDKW epitopes. The HIV immunogen of the invention can be part of gp160or gp120. Thus the HIV immunogens ELKDKWA or LDKWA, for example, can bea part of a region of go120 or a region of gp160; for instance, part ofgp120V3. Accordingly, the invention comprehends a gp120 or gp160modified so as to contain an epitope not naturally occurring in gp160.The epitope can be a B-cell epitope. The epitope, more specifically, canbe at least one of HIV1gag(+pro) (IIIB), gp120(MN) (+transmembrane),nef(BRU)CTL, pol(IIIB)CTL, and ELDKWA or LDKW epitopes. The gp120 can bemodified in the V3 loop. The immunogen and modified gp120 or gp160 canbe synthesized by any suitable vector, including a poxvirus, such as arecombinant of the invention; or, by any suitable chemical synthesismethod such as the Merrifield Synthesis Method.

The invention in yet a further aspect relates to the product ofexpression of the inventive recombinant poxvirus and uses therefor, aswell as to uses for the inventive immunogens and modified gp120 andsp160, such as to form antigenic, immunological or vaccine compositionsfor treatment, prevention, diagnosis or testing. The invention in stilla further embodiment relates to the uses of DNA from the recombinants asprobes for detecting the presence or absence of HIV DNA in a sample orfor DNA immunization using an appropriate expression plasmid.

These and other embodiments are disclosed or are obvious from andencompassed by the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings, in which:

FIG. 1 schematically shows a method for the construction of plasmidpSD460 for deletion of thymidine kinase gene and generation ofrecombinant vaccinia virus vP410;

FIG. 2 schematically shows a method for the construction of plasmidpSD486 for deletion of hemorrhagic region and generation of recombinantvaccinia virus vP553;

FIG. 3 schematically shows a method for the construction of plasmidpMP494Δ for deletion of ATI region and generation of recombinantvaccinia virus vP618;

FIG. 4 schematically shows a method for the construction of plasmidpSD467 for deletion of hemagglutinin gene and generation of recombinantvaccinia virus vP723;

FIG. 5 schematically shows a method for the construction of plasmidpMPCK1Δ for deletion of gene cluster [C7L-K1L] and generation ofrecombinant vaccinia virus vP804;

FIG. 6 schematically shows a method for the construction of plasmidpSD548 for deletion of large subunit, ribonucleotide reductase andgeneration of recombinant vaccinia virus vP866 (NYVAC);

FIG. 7 schematically shows a method for the construction of plasmidpRW842 for insertion of rabies glycoprotein G gene into the TK deletionlocus and generation of recombinant vaccinia virus vP879;

FIG. 8 shows the DNA sequence (SEQ ID NO:66) of a canarypox PvuIIfragment containing the C5 ORF.

FIGS. 9A and 9B schematically show a method for the construction ofrecombinant canarypox virus vCP65 (ALVAC-RG);

FIG. 10 shows schematically the ORFs deleted to generate NYVAC;

FIG. 11 shows the nucleotide sequence (SEQ ID NO:67) of a fragment ofTROVAC DNA containing an F8 ORF;

FIG. 12 shows the DNA sequence (SEQ ID NO:68) of a 2356 base pairfragment of TROVAC DNA containing the F7 ORF;

FIGS. 13A to 13D show graphs of rabies neutralizing antibody titers(RFFIT, IU/ml), booster effect of HDC and vCP65 (10^(5.5) TCID₅₀) involunteers previously immunized with either the same or the alternatevaccine (vaccines given at days 0, 28 and 180, antibody titers measuredat days 0, 7, 28, 35, 56, 173, 187 and 208);

FIG. 14A to 14C shows the nucleotide sequence of the H6-promoted HIV1gp120 (+transmembrane) gene and the I3L-promoted HIV1gag(+pro) genecontained in pHIV32 (SEQ ID NOS: 78 and 79);

FIG. 15A to 15F shows the nucleotide sequence of the C3 locus inpVQH6CP3L (SEQ ID NOS: 80 and 81);

FIG. 16 shows the nucleotide sequence of the I3L-promoted nef CTL2epitope and H6-promoted nef CTL1 epitope contained in p2-60-HIV.3 (SEQID NOS: 93-96);

FIG. 17A to 17C shows the nucleotide sequence of the C6 locus in pC6L(SEQ ID NOS: 97 and 98);

FIG. 18A to 18B shows the nucleotide sequence of the I3L-promoted pol2epitope, H6-promoted poll epitope and 42K-promoted pol3 epitopecontained in pC5POLT5A (SEQ ID NOS: 111-115);

FIG. 19A to 19C shows the nucleotide sequence of the C5 locus inpNC5L-SP5 (SEQ ID NOS: 116 and 117);

FIG. 20 shows the rabbit antibody responses to the HIV envelopeglycoprotein following immunization with ALVAC, vCP205, or with peptideCLTB-36;

FIG. 21 shows the rabbit antibody responses to the HIV MN V3 loopfollowing immunization with ALVAC, vCP205, or with peptide CLTB-36;

FIG. 22 shows the guinea pig antibody responses to the HIV envelopeglycoprotein following immunization with ALVAC, vCP205, or with peptideCLTB-36;

FIG. 23 shows the guinea pig antibody responses to the HIV MN V3 loopfollowing immunization with ALVAC, vCP205, or with peptide CLTB-36;

FIG. 24 shows in vitro stimulation of HIV-1-specific CTLs from PBMCs ofan HIV-seropositive individual—Patient 1;

FIG. 25 is as in FIG. 24 but with Patient 2;

FIG. 26 a-c, shows the nucleotide sequence of the H6-promoted HIV1gp120+TM (with ELDKWA epitopes) gene (SEQ ID NOS: 135 and 136) containedin pHIV59 and vCP1307 and the protein expressed (SEQ ID NO: 137);

FIG. 27 shows FACS analysis of vCP1307-infected cells (FACS analysis wasperformed on HeLa cells infected with ALVAC, vP1286 or vCP1307 with serafrom HIV1-seropositve humans (upper panel) or a human monoclonalantibody specific for the ELDKWA epitope, IAM41-2F5 (lower panel));

FIG. 28 a-c shows the nucleotide sequence of the H6-promoted HIV1gp120+TM (with ELDKWA epitopes) gene (SEQ ID NOS: 138 and 139) containedin pHIV60 and vP1313 and the protein expressed (SEQ ID NO: 140);

FIG. 29 shows FACS analysis of vP1313-infected cells (FACS analysis wasperformed on HeLa cells infected with NYVAC, vP1286 or vP1313 with serafrom HIV1-seropositve humans (upper panel) or a human monoclonalantibody specific for the ELDKWA epitope, IAM41-2F5 (lower panel)).

FIG. 30 a-c shows the nucleotide sequence of the H6-promoted HIV1gp120+TM (with ELDKWA epitopes) gene (SEQ ID NOS: 141 to 142) containedin pHIV61 and vP1319 and the protein expressed (SEQ ID NO: 143);

FIG. 31 shows the FACS analysis of vP1319-infected cells (FACS analysiswas performed on HeLa cells infected with WR, vP1286 or vP1319 with serafrom HIV1-seropositve humans (upper panel), a human monoclonal antibodyspecific for the ELDKWA epitope, IAM41-2F5 (middle panel) or a mousemonoclonal antibody specific for the V3 loop, 50.1 (lower panel));

FIGS. 32, 33 a, 33 b, 33 c, 34, 35, 36, 37 a, 37 b, 37 c, 38 a, 38 b, 38c show comparative body weights (FIG. 32), blood counts (FIG. 33 a-c),creatinine (FIG. 34), SGOT (35), SGPT (FIG. 36), ELISA (Anti-gp160MN/BR, -v3MN, -p24, FIGS. 37 a-c, 38 a-c) of monkeys inoculated withvCP205 and placebo (FIG. 32). upper panel=monkeys 1-4, placebo; lowerpanel=monkeys 5-8 vCP205; monkeys: 1=open square, 2=open diamond, 3=opentriangle, 4=open circle, 5=darkened square, 6=darkened diamond,7=darkened triangle, 8—darkened circle; plots of Kg (wt) vs. weeks(inoculations indicated with arrow). FIG. 33 a: leucoytes: left top andbottom panels=monkeys 1-4, placebo; right top and bottom panels=monkeys5-8, vCP205; top panels individual WBC counts, key same as FIG. 32except small darkened circle is mean (m); lower panels differential cellcounts, darkened square=granulo, open square=lympho, darkeneddiamond=mono. FIG. 33 b: same layout and keying as FIG. 33 a, with upperpanels indicating erythrocytes and lower panels indicating meancorpuscle volume and mean indicated by smaller darkened circle. FIG. 33c: same layout as FIG. 33 b with upper panels indicating hematocrite andlower panels indicating hemoglobin. FIG. 34: upper bar graphs=monkeys1-4, placebo; lower bar graph=monkeys 5-8, vCP205; mg/l vs. days, arrowindicates inoculation; monkeys 1 and 5=dark bars, monkeys 2 and 6=doublestippling bars (slanted lines in opposite directions), monkeys 3 and7=dotted bars, monkeys 4 and 8=single stippling bar (slant lines in onedirection), mean is darkened circles. FIG. 35: same keying as FIG. 34,except IU/l vs. days. FIG. 36: same keying as FIG. 35. FIGS. 37 a-c and38 a-c: ELISA in placebo administered monkeys (FIGS. 37 a-c) and invCP205 administered monkeys (FIGS. 38 a-c), titer (log) vs. weeks, arrowindicates injection; FIGS. 37 a and 38 a=anti-gp160 MN/BRU, FIGS. 37 band 38 b=anti-V3MN, FIGS. 37 c and 38C=anti-p24; monkeys 1 and 5=opencircle; monkeys 2 and 6=darkened circle; monkeys 3 and 7=open invertedtriangle; monkeys 4 and 8=darkened inverted triangle);

FIG. 39 shows anti-HIV1 (MN) neutralizing antibodies in monkeysinoculated with vCP205 (keying same as FIG. 38 a-c); and,

FIGS. 40, 41 a, 41 b, 41 c, 42, 43 a, 43 b, 43 c, 43 d, 44 a, 44 b, 45a, 45 b, 46 a, 46 b, 47 a, and 47 b show leucocyte counts (FIG. 40),blood counts (erythrocytes FIG. 41 a, hematocrite FIG. 41 b,reticulocytes FIG. 41 c), prothrombin (FIG. 42), biochemical results(total cholesterol, total proteins, glucose FIG. 43 a; sodium, potassiumFIG. 43 b; creatinine, bilirubin FIG. 43 c; SGOTransaminase,SGPTransaminase, alkaline phosphatase FIG. 43 d), gp160 MN/BRU ELISA(control FIG. 44 a, test animals FIG. 44 b), V3 MN ELISA (control FIG.46 a, test animals FIG. 46 b), and nef ELISA (control FIG. 47 a, testanimals FIG. 47 b) in monkeys inoculated with vCP300 and a placebo (FIG.40: layout same as FIG. 33 a; keying same as FIG. 33 a, except in upperpanels, mean is dotted circle (left) and open circle (right) and inlower panels decimal instead of percentage and darkened square=neutro,open diamond=eosino, and darkened triangle=baso. FIG. 41 a: layout sameas FIG. 33 b, keying same as FIG. 33 a, except mean is dotted circle(left) and open circle (right). FIG. 41 b: layout same as FIG. 33 c,keying same as FIG. 41 a. FIG. 41 c: layout and keying same as FIG. 41b, upper panels=reticulocytes, lower panels=thrombocytes. FIG. 42: upperpanel=placebo, lower panel=vCP300, keying same as FIG. 41 c. FIG. 43 a:top=cholesterol, middle=proteins, lower=glucose; open circle=placebo,darkened circle=vCP300. FIG. 34 b: top=sodium, lower=potassium; keyingsame as FIG. 43 a. FIG. 43 c: top=creatinine, lower=bilirulain; keyingsame as FIG. 43 a. FIG. 43 d: top=SGOT, middle=SGPT, lower=alkalinephosphatases; keying same as FIG. 43 a. FIGS. 44 a, 44 b: gp160 MN/BRUELISA, control and vCP300, respectively; keying same as FIGS. 37 a and38 a, respectively. FIGS. 45 a, 45 b: V3 MN ELISA, control and vCP300,respectively; keying same as FIGS. 37 b and 38 b, respectively. FIGS. 46a, 46 b: p34 ELISA, control and vCP300, respectively; keying same asFIGS. 37 c and 38 c, respectively. FIG. 47 a, 47 b, nef ELISA, controland vCP300, respectively; keying as in FIGS. 44 a-46 b).

DETAILED DESCRIPTION OF THE INVENTION

To develop a new vaccinia vaccine strain, NYVAC (vP866), the Copenhagenvaccine strain of vaccinia virus was modified by the deletion of sixnonessential regions of the genome encoding known or potential virulencefactors. The sequential deletions are detailed below. All designationsof vaccinia restriction fragments, open reading frames and nucleotidepositions are based on the terminology reported in Goebel et al.,1990a,b.

The deletion loci were also engineered as recipient loci for theinsertion of foreign genes.

The regions deleted in NYVAC are listed below. Also listed are theabbreviations and open reading frame designations for the deletedregions (Goebel et al., 1990a,b) and the designation of the vacciniarecombinant (vP) containing all deletions through the deletionspecified:

-   -   (1) thymidine kinase gene (TK; J2R) vP410;    -   (2) hemorrhagic region (u; B13R+B14R) vP553;    -   (3) A type inclusion body region (ATI; A26L) vP618;    -   (4) hemagglutinin gene (HA; A56R) vP723;    -   (5) host range gene region (C7L-K1L) vP804; and    -   (6) large subunit, ribonucleotide reductase (I4L) vP866 (NYVAC).

NYVAC is a genetically engineered vaccinia virus strain that wasgenerated by the specific deletion of eighteen open reading framesencoding gene products associated with virulence and host range. NYVACis highly attenuated by a number of criteria including i) decreasedvirulence after intracerebral inoculation in newborn mice, ii) inocuityin genetically (nu⁺/nu⁺) or chemically (cyclophosphamide)immunocompromised mice, iii) failure to cause disseminated infection inimmunocompromised mice, iv) lack of significant induration andulceration on rabbit skin, v) rapid clearance from the site ofinoculation, and vi) greatly reduced replication competency on a numberof tissue culture cell lines including those of human origin.Nevertheless, NYVAC based vectors induce excellent responses toextrinsic immunogens and provided protective immunity.

TROVAC refers to an attenuated fowlpox that was a plaque-cloned isolatederived from the FP-1 vaccine strain of fowlpoxvirus which is licensedfor vaccination of 1 day old chicks. ALVAC is an attenuated canarypoxvirus-based vector that was a plaque-cloned derivative of the licensedcanarypox vaccine, Kanapox (Tartaglia et al., 1992). ALVAC has somegeneral properties which are the same as some general properties ofKanapox. ALVAC-based recombinant viruses expressing extrinsic immunogenshave also been demonstrated efficacious as vaccine vectors (Tartaglia etal., 1993 a,b). This avipox vector is restricted to avian species forproductive replication. On human cell cultures, canarypox virusreplication is aborted early in the viral replication cycle prior toviral DNA synthesis. Nevertheless, when engineered to express extrinsicimmunogens, authentic expression and processing is observed in vitro inmammalian cells and inoculation into numerous mammalian species inducesantibody and cellular immune responses to the extrinsic immunogen andprovides protection against challenge with the cognate pathogen (Tayloret al., 1992; Taylor et al., 1991). Recent Phase I clinical trials inboth Europe and the United States of a canarypox/rabies glycoproteinrecombinant (ALVAC-RG) demonstrated that the experimental vaccine waswell tolerated and induced protective levels of rabiesvirus neutralizingantibody titers (Cadoz et al., 1992; Fries et al., 1992). Additionally,peripheral blood mononuclear cells (PBMCs) derived from the ALVAC-RGvaccinates demonstrated significant levels of lymphocyte proliferationwhen stimulated with purified rabies virus (Fries et al., 1992).

NYVAC, ALVAC and TROVAC have also been recognized as unique among allpoxviruses in that the National Institutes of Health (“NIH”) (U.S.Public Health Service), Recombinant DNA Advisory Committee, which issuesguidelines for the physical containment of genetic material such asviruses and vectors, i.e., guidelines for safety procedures for the useof such viruses and vectors which are based upon the pathogenicity ofthe particular virus or vector, granted a reduction in physicalcontainment level: from BSL2 to BSL1. No other poxvirus has a BSL1physical containment level. Even the Copenhagen strain of vacciniavirus—the common smallpox vaccine—has a higher physical containmentlevel; namely, BSL2. Accordingly, the art has recognized that NYVAC,ALVAC and TROVAC have a lower pathogenicity than any other poxvirus.

Both NYVAC- and ALVAC-based recombinant viruses have been shown tostimulate in vitro specific CD8⁺ CTLs from human PBMCs (Tartaglia etal., 1993a). Mice immunized with NYVAC or ALVAC recombinants expressingvarious forms of the HIV-1 envelope glycoprotein generated both primaryand memory HIV specific CTL responses which could be recalled by asecond inoculation (Tartaglia et al., 1993a; Cox et al., 1993).ALVAC-env and NYVAC-env recombinants (expressing the HIV-1 envelopeglycoprotein) stimulated strong HIV-specific CTL responses fromperipheral blood mononuclear cells (PBMC) of HIV-1 infected individuals(Tartaglia et al., 1993a; Cox et al., 1993). Acutely infected autologousPBMC were used as stimulator cells for the remaining PBMC. After 10 daysincubation in the absence of exogenous IL-2, the cells were evaluatedfor CTL activities. NYVAC-env and ALVAC-env stimulated high levels ofanti-HIV activities in mice.

Applicants have generated an ALVAC recombinant, vCP300(ALVAC-MN120TMGNP), that expresses numerous HIV1 antigens and HIV1T-cell epitopes. vCP300 expresses the HIV1 (IIIB) gag (and protease)proteins. (Expression of the protease protein allows the gag polyproteinto be correctly processed.) vCP300 also expresses a form of the HIV1(MN) envelope glycoprotein in which gp120 is fused to the transmembraneanchor sequence derived from gp41. vP300 also expresses two (2) HIV1(BRU) nef CTL epitopes and three (3) HIV1 (IIIB) pol CTL epitopes.vCP300 does not, however, express a functional reverse transcriptaseactivity. vCP300 also does not express a functional nef gene product; aprotein associated with pathogenicity in the SIV-macaque model systemand HIV1 virulence (Miller et al, 1994; Spina et al, 1994). Therefore,vCP300 expresses immunologically important antigens and/or epitopes fromgag, env, pol and nef, but does not express the potentially detrimentalenzymatic and/or pathogenic activities associated with pol and nef.

As previously mentioned, vCP300 expresses a form of HIV1 envelopeglycoprotein in which the vast majority of the gp41 sequence is deleted.Since most of the immunologically important epitopes associated with theHIV1 envelope glycoprotein are found on gp120, rather than gp41, it isassumed that the immunogenicity of the envelope glycoprotein expressedby this recombinant is not adversely affected. In fact, in aside-by-side analysis, an HIV1 gp120 subunit vaccine was able to protectchimpanzees against an HIV1 challenge, whereas an HIV1 gp160 subunitvaccine was not (Berman et al., 1990). It is not known why the efficacyof these two vaccines is different. However, it is known that antibodiesagainst an epitope gp41 can enhance HIV1 infection in vitro (Robinson etal., 1990). Furthermore, it is known that antibodies to a putativeimmunosuppressive region of gp41 are associated with the absence of AIDSin HIV1-seropositive individuals, suggesting a potential role inpathogenicity for this region (Klasse et al., 1988). In addition, it isknown that antibodies to the C-terminal region of gp41 can cross-reactwith HLA class II antigens (Golding et al., 1988) and inhibitantigen-specific lymphoproliferative responses (Golding et al., 1989).Since the envelope glycoprotein expressed by vCP300 does not contain anygp41 sequence, except for the 28 amino acids associated with thetransmembrane region, the potentially detrimental effects associatedwith gp41 are avoided. Furthermore, the envelope glycoprotein expressedby vCP300 does not contain the immunodominant epitope on gp41 that isrecognized by antisera from every HIV1-seropositive individual fromevery stage of an HIV1 infection (Shafferman et al., 1989). Therefore,diagnostic tests based upon reactivity against this epitope can be usedto distinguish between vaccinated and infected individuals. The abilityto differentiate vCP300-vaccinated individuals from HIV1-infectedindividuals with a gp41 antibody assay is important because the mostcommonly used diagnostic kit (which assays for the presence of HIV1 p24antibodies) would be useless, since vCP300-vaccinated individuals wouldbe expected to have a high level of p24 antibodies. Alternatively, HIV-1infected individuals would be expected to make anti-gp41 antibodies butthose vaccinated with vCP205 or vCP300 would not since gp41 is absentfrom vCP205 or vCP300.

Rabbits and guinea pigs have been inoculated with an ALVAC recombinant(vCP205; ALVAC-MN120TMG) expressing the same cell surface-associatedform of HIV1 gp120 (120™) and Gag/pro as expressed by vCP300. Rabbitsand guinea pigs have also been inoculated with vCP205 and boosted withan HIV1 T-B peptide. Both ALVAC-based protocols were able to elicit HIV1gp160- and V3 loop-specific antibodies, thereby indicating that an ALVACrecombinant expressing the cell surface form of HIV1 gp120 induces anHIV1-specific immune response.

vCP300 expresses the HIV1 Gag proteins, a cell surface-associated formof the HIV1 gp120 envelope glycoprotein, two (2) regions from HIV1 nefcontaining CTL epitopes and three (3) regions from HIV1 pol containingCTL epitopes. The expression of an HIV1 envelope glycoprotein that doesnot contain gp41 allows vaccinated individuals to be differentiated fromHIV1-infected individuals via an assay for gp41 antibodies andeliminates potentially detrimental responses associated with variousgp41 epitopes. Since a previous ALVAC recombinant expressing HIV1 gp160has been shown to elicit HIV1-specific humoral and cellular immuneresponses in humans (Pialoux et al., 1995), the addition of Gag and thePol and Nef epitopes (and the deletion of the potentially detrimentalgp41 epitopes) heightens and broadens the immune response elicited byvP300, relative to vCP125, and, may provide an efficacious HIV1 vaccine,or immunological or antigenic composition.

In Macaca fascicularis (monkeys; macaques) immunized with vCP205 orvCP300, an antibody response (anti-HIV) was observed, thereby furtherdemonstrating the utility and efficacy of these recombinants.

Since the ELDKWA or LDKW epitope does not appear to be very immunogenicin its normal configuration, to increase its immunogenicity,recombinants of the invention present it to the immune system in a moreimmunogenic setting, such as within the V3 loop of gp120 or within otherregions of gp120 and/or as part of an intact gp160 envelope.

ALVAC recombinant (vCP1307), NYVAC recombinant (vP1313) and COPAKrecombinant (vP1319) express a form of the HIV1 gp120+TM gene product inwhich the V3 loop has been modified to contain two copies of the ELDKWAepitope. The ELDKWA epitopes of this gp120+TM (with ELDKWA epitopes)gene product are expressed on the surface of vCP1307-, vP1313- andvP1319-infected cells.

The V3 loop of HIV1 gp120+TM (or gp160) can be used as an immunologicalplatform for any linear epitope, not just linear HIV1 epitopes. Thegp120+TM (with epitopes of interest) protein generated by theserecombinants can also be isolated from poxvirus-infected cells and usedto inoculate individuals in a subunit vaccine configuration(composition, or an antigenic or immunological composition). Theproteins generated by the recombinants and antibodies elicited therefromcan also be used in assays to detect the presence or absence of HIV.Accordingly, the invention comprehends HIV immunogens and modified gp120and gp160. Further, such envelope-based immunogens (HIV immunogens orunmodified gp120 or gp160 (can be derived from any eukaryotic orprokaryotic expression vector and used as subunit preparations or can beadministered through DNA immunization using an appropriate expressionplasmid. Techniques for DNA immunization are known in the art. Withrespect to techniques for DNA immunization, mention is particularly madeof Nabel and Felgner, “Direct gene transfer for immunotherapy andimmunization”, Tibtech, May 1993, 11; 211-215, and Webster et al,“protection of ferrets against influenza challenge with a DNA vaccine tothe haemagglutinin”, vaccine, 1994, 12(16): 1495-1498, incorporatedherein by reference. Also, the DNA from the recombinants vP1313, vP1319and vCP1307 can be used to probe for the presence of HIV DNA in a sampleof interest using known hybridization techniques, or, to generate PCRprimers using known techniques.

Clearly based on the attenuation profiles of the NYVAC, ALVAC, andTROVAC vectors and their demonstrated ability to elicit both humoral andcellular immunological responses to extrinsic immunogens (Tartaglia etal., 1993a,b; Taylor et al., 1992; Konishi et al., 1992) suchrecombinant viruses offer a distinct advantage over previously describedvaccinia-based recombinant viruses.

The administration procedure for recombinant virus, immunogen, modifiedgp120 or gp160, DNA or expression product compositions of the inventionsuch as immunological, antigenic or vaccine compositions or therapeuticcompositions can be via a parenteral route (intradermal, intramuscularor subcutaneous). Such an administration enables a systemic immuneresponse.

More generally, the inventive antigenic, immunological or vaccinecompositions or therapeutic compositions (compositions containing thepoxvirus recombinants, expression products, immunogens, DNA, modifiedgp120 or gp160 of the invention) can be prepared in accordance withstandard techniques well known to those skilled in the pharmaceuticalart. Such compositions can be administered in dosages and by techniqueswell known to those skilled in the medical arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular patient, and the route of administration. The compositionscan be administered alone, or can be co-administered or sequentiallyadministered with compositions of the invention or with otherimmunological, antigenic or vaccine or therapeutic compositions inseropositive individuals. The compositions can be administered alone, orcan be co-administered or sequentially administered with compositions ofthe invention or with other antigenic, immunological, vaccine ortherapeutic compositions in seronegative individuals. Such othercompositions can include purified antigens from immunodeficiency virusor from the expression of such antigens by a recombinant poxvirus orother vector system or, such other compositions can include arecombinant poxvirus which expresses other immunodeficiency antigens orbiological response modifiers (e.g. cytokines; co-stimulatingmolecules). Again, co-administration is performed by taking intoconsideration such known factors as the age, sex, weight, and conditionof the particular patient, and, the route of administration.

Examples of compositions of the invention include liquid preparationsfor orifice, e.g., oral, nasal, anal, vaginal, etc., administration suchas suspensions, syrups or elixirs; and, preparations for parenteral,subcutaneous, intradermal, intramuscular or intravenous administration(e.g., injectable administration) such as sterile suspensions oremulsions. In such compositions the recombinant poxvirus, expressionproduct, immunogen, DNA, or modified gp120 or gp160 may be in admixturewith a suitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose or the like.

Further, the products of expression of the inventive recombinantpoxviruses can be used directly to stimulate an immune response ineither seronegative or seropositive individuals or in animals. Thus, theexpression products can be used in compositions of the invention insteador in addition to the inventive recombinant poxvirus in theaforementioned compositions. The immunogens of the invention can besimilarly used.

Additionally, the inventive recombinant poxvirus and the expressionproducts therefrom and immunogens and modified gp120 or gp160 of theinvention stimulate an immune or antibody response in humans andanimals. From those antibodies or by techniques well-known in the art,monoclonal antibodies can be prepared and, those monoclonal antibodies,can be employed in well known antibody binding assays, diagnostic kitsor tests to determine the presence or absence of particularimmunodeficiency virus antigen(s) and therefore the presence or absenceof the virus, or to determine whether an immune response to the virus orantigen(s) has simply been stimulated. Those monoclonal antibodies canalso be employed in immunoadsorption chromatography to recoverimmunodeficiency virus or expression products of the inventiverecombinant poxvirus.

Monoclonal antibodies are immunoglobulins produced by hybridoma cells. Amonoclonal antibody reacts with a single antigenic determinant andprovides greater specificity than a conventional, serum-derivedantibody. Furthermore, screening a large number of monoclonal antibodiesmakes it possible to select an individual antibody with desiredspecificity, avidity and isotype. Hybridoma cell lines provide aconstant, inexpensive source of chemically identical antibodies andpreparations of such antibodies can be easily standardized. Methods forproducing monoclonal antibodies are well known to those of ordinaryskill in the art, e.g., Koprowski, H. et al., U.S. Pat. No. 4,196,265,issued Apr. 1, 1989, incorporated herein by reference.

Uses of monoclonal antibodies are known. One such use is in diagnosticmethods, e.g., David, G. and Greene, H. U.S. Pat. No. 4,376,110, issuedMar. 8, 1983; incorporated herein by reference. Monoclonal antibodieshave also been used to recover materials by immunoadsorptionchromatography, e.g., Milstein, C. 1980, Scientific American 243:66, 70,incorporated herein by reference.

Furthermore, the inventive recombinant poxvirus or expression productstherefrom or the inventive immunogens or modified gp120 or gp160 can beused to stimulate a response in cells such as lymphocytes or CTLs invitro or ex vivo for subsequent reinfusion into a patient. If thepatient is seronegative, the reinfusion is to stimulate an immuneresponse, e.g., an immunological or antigenic response such as activeimmunization. In a seropositive individual, the reinfusion is tostimulate or boost the immune system against immunodeficiency virus.

Additionally, the DNA from inventive recombinants can be used as probesto detect the presence of HIV DNA in a sample or, to generate PCRprimers, or for DNA immunization using an appropriate expressionplasmid, by methods known in the art. (See Nabel and Felger and Websteret al, Supra)

Accordingly, the inventive recombinant poxvirus has several utilities:In antigenic, immunological or vaccine compositions such as foradministration to seronegative individuals. In therapeutic compositionsin seropositive individuals in need of therapy to stimulate or boost theimmune system against immunodeficiency virus. In vitro to produceantigens or the inventive immunogens or the inventive modified gp120 orgp160 which can be further used in antigenic, immunological or vaccinecompositions or in therapeutic compositions. To generate antibodies(either by direct administration or by administration of an expressionproduct of the inventive recombinant poxvirus) which can be furtherused: in diagnosis, tests or kits to ascertain the presence or absenceof antigens in a sample such as sera, for instance, to ascertain thepresence or absence of immunodeficiency virus or CTLs in a sample suchas sera or, to determine whether an immune response has elicited to thevirus or, to particular antigen(s); or, in immunoadsorptionchromatography (the inventive immunogens and modified gp120 or gp160 canalso be used to generate antibodies which can be also so further used).To generate DNA for use as hybridization probes or to prepare PCRprimers or for DNA immunization. And, the inventive recombinantpoxvirus, expression products therefrom, immunogens and modified gp120or gp160 can be used to generate stimulated cells which can be furtherused (reinfused) to stimulate an immune response (antigenic, orimmunological response; or active immunization) or, to boost orstimulate the immune system (for instance, of an immunocompromised orseropositive individual). Other utilities also exist for embodiments ofthe invention.

A better understanding of the present invention and of its manyadvantages will be had from the following examples, given by way ofillustration.

EXAMPLES

DNA Cloning and Synthesis. Plasmids were constructed, screened and grownby standard procedures (Maniatis et al., 1982; Perkus et al., 1985;Piccini et al., 1987). Restriction endonucleases were obtained fromBethesda Research Laboratories, Gaithersburg, Md., New England Biolabs,Beverly, Mass.; and Boehringer Mannheim Biochemicals, Indianapolis, Ind.Klenow fragment of E. coli polymerase was obtained from BoehringerMannheim Biochemicals. BAL-31 exonuclease and phage T4 DNA ligase wereobtained from New England Biolabs. The reagents were used as specifiedby the various suppliers.

Synthetic oligodeoxyribonucleotides were prepared on a Biosearch 8750 orApplied Biosystems 380B DNA synthesizer as previously described (Perkuset al., 1989). DNA sequencing was performed by the dideoxy-chaintermination method (Sanger et al., 1977) using Sequenase (Tabor et al.,1987) as previously described (Guo et al., 1989). DNA amplification bypolymerase chain reaction (PCR) for sequence verification (Engelke etal., 1988) was performed using custom synthesized oligonucleotideprimers and GeneAmp DNA amplification Reagent Kit (Perkin Elmer Cetus,Norwalk, Conn.) in an automated Perkin Elmer Cetus DNA Thermal Cycler.Excess DNA sequences were deleted from plasmids by restrictionendonuclease digestion followed by limited digestion by BAL-31exonuclease and mutagenesis (Mandecki, 1986) using syntheticoligonucleotides.

Cells, Virus, and Transfection. The origins and conditions ofcultivation of the Copenhagen strain of vaccinia virus has beenpreviously described (Guo et al., 1989). Generation of recombinant virusby recombination, in situ hybridization of nitrocellulose filters andscreening for B-galactosidase activity are as previously described(Piccini et al., 1987).

The origins and conditions of cultivation of the Copenhagen strain ofvaccinia virus and NYVAC has been previously described (Guo et al.,1989; Tartaglia et al., 1992). Generation of recombinant virus byrecombination, in situ hybridization of nitrocellulose filters andscreening for B-galactosidase activity are as previously described(Panicali et al., 1982; Perkus et al., 1989).

The parental canarypox virus (Rentschler strain) is a vaccinal strainfor canaries. The vaccine strain was obtained from a wild type isolateand attenuated through more than 200 serial passages on chick embryofibroblasts. A master viral seed was subjected to four successive plaquepurifications under agar and one plaque clone was amplified through fiveadditional passages after which the stock virus was used as the parentalvirus in in vitro recombination tests. The plaque purified canarypoxisolate is designated ALVAC.

The strain of fowlpox virus (FPV) designated FP-1 has been describedpreviously (Taylor et al., 1988a). It is an attenuated vaccine strainuseful in vaccination of day old chickens. The parental virus strainDuvette was obtained in France as a fowlpox scab from a chicken. Thevirus was attenuated by approximately 50 serial passages in chickenembryonated eggs followed by 25 passages on chicken embryo fibroblastcells. The virus was subjected to four successive plaque purifications.One plaque isolate was further amplified in primary CEF cells and astock virus, designated as TROVAC, established.

NYVAC, ALVAC and TROVAC viral vectors and their derivatives werepropagated as described previously (Piccini et al., 1987; Taylor et al.,1988a,b). Vero cells and chick embryo fibroblasts (CEF) were propagatedas described previously (Taylor et al., 1988a,b).

Example 1 Construction of Plasmid pSD460 for Deletion of ThymidineKinase Gene (J2R)

Referring now to FIG. 1, plasmid pSD406 contains vaccinia HindIII J(pos. 83359-88377) cloned into pUC8. pSD406 was cut with HindIII andPvuII, and the 1.7 kb fragment from the left side of HindIII J clonedinto pUC8 cut with HindIII/SmaI, forming pSD447. pSD447 contains theentire gene for J2R (pos. 83855-84385). The initiation codon iscontained within an NlaIII site and the termination codon is containedwithin an SspI site. Direction of transcription is indicated by an arrowin FIG. 1.

To obtain a left flanking arm, a 0.8 kb HindIII/EcoRI fragment wasisolated from pSD447, then digested with NlaIII and a 0.5 kbHindIII/NlaIII fragment isolated. Annealed synthetic oligonucleotidesMPSYN43/MPSYN44 (SEQ ID NO:1/SEQ ID NO:2)

                     SmaI MPSYN43 5′     TAATTAACTAGCTACCCGGG     3′MPSYN44 3′ GTACATTAATTGATCGATGGGCCCTTAA 5′  NlaIII                  EcoRIwere ligated with the 0.5 kb HindIII/NlaIII fragment into pUC18 vectorplasmid cut with HindIII/EcoRI, generating plasmid pSD449.

To obtain a restriction fragment containing a vaccinia right flankingarm and pUC vector sequences, pSD447 was cut with SspI (partial) withinvaccinia sequences and HindIII at the pUC/vaccinia junction, and a 2.9kb vector fragment isolated. This vector fragment was ligated withannealed synthetic oligonucleotides MPSYN45/MPSYN46 (SEQ ID NO:3/SEQ IDNO:4)

  HindIII SmaI MPSYN45 5′  AGCTTCCCGGGTAAGTAATACGTCAAGGAGAAAACGAAMPSYN46 3′      AGGGCCCATTCATTATGCAGTTCCTCTTTTGCTT               NotI          SspI ACGATCTGTAGTTAGCGGCCGCCTAATTAACTAAT  3′ MPSYN45 TGCTAGACATCAATCGCCGGCGGATTAATTGATTA  5′ MPSYN46generating pSD459.

To combine the left and right flanking arms into one plasmid, a 0.5 kbHindIII/SmaI fragment was isolated from pSD449 and ligated with pSD459vector plasmid cut with HindIII/SmaI, generating plasmid pSD460. pSD460was used as donor plasmid for recombination with wild type parentalvaccinia virus Copenhagen strain VC-2. ³²P labelled probe wassynthesized by primer extension using MPSYN45 (SEQ ID NO:3) as templateand the complementary 20mer oligonucleotide MPSYN47 (SEQ ID NO:5) (5′TTAGTTAATTAGGCGGCCGC 3′) as primer. Recombinant virus vP410 wasidentified by plaque hybridization.

Example 2 Construction of Plasmid pSD486 for Deletion of HemorrhagicRegion (B13R+B14R)

Referring now to FIG. 2, plasmid pSD419 contains vaccinia SalI G (pos.160, 744-173,351) cloned into pUC8. pSD422 contains the contiguousvaccinia SalI fragment to the right, SalI J (pos. 173, 351-182,746)cloned into pUC8. To construct a plasmid deleted for the hemorrhagicregion, u, B13R-B14R (pos. 172, 549-173,552), pSD419 was used as thesource for the left flanking arm and pSD422 was used as the source ofthe right flanking arm. The direction of transcription for the u regionis indicated by an arrow in FIG. 2.

To remove unwanted sequences from pSD419, sequences to the left of theNcoI site (pos. 172,253) were removed by digestion of pSD419 withNcoI/SmaI followed by blunt ending with Klenow fragment of E. colipolymerase and ligation generating plasmid pSD476. A vaccinia rightflanking arm was obtained by digestion of pSD422 with HpaI at thetermination codon of B14R and by digestion with NruI 0.3 kb to theright. This 0.3 kb fragment was isolated and ligated with a 3.4 kbHincII vector fragment isolated from pSD476, generating plasmid pSD477.The location of the partial deletion of the vaccinia u region in pSD477is indicated by a triangle. The remaining B13R coding sequences inpSD477 were removed by digestion with ClaI/HpaI, and the resultingvector fragment was ligated with annealed synthetic oligonucleotidesSD22mer/SD20mer (SEQ ID NO:6/SEQ ID NO:7)

    ClaI         BamHI HpaI SD22mer 5′  CGATTACTATGAAGGATCCGTT  3′SD20mer 3′    TAATGATACTTCCTAGGCAA  5′generating pSD479. pSD479 contains an initiation codon (underlined)followed by a BamHI site. To place E. coli Beta-galactosidase in theB13-B14 (u) deletion locus under the control of the u promoter, a 3.2 kbBamHI fragment containing the Beta-galactosidase gene (Shapira et al.,1983) was inserted into the BamHI site of pSD479, generating pSD479BG.pSD479BG was used as donor plasmid for recombination with vaccinia virusvP410. Recombinant vaccinia virus vP533 was isolated as a blue plaque inthe presence of chromogenic substrate X-gal. In vP533 the B13R-B14Rregion is deleted and is replaced by Beta-galactosidase.

To remove Beta-galactosidase sequences from vP533, plasmid pSD486, aderivative of pSD477 containing a polylinker region but no initiationcodon at the u deletion junction, was utilized. First the ClaI/HpaIvector fragment from pSD477 referred to above was ligated with annealedsynthetic oligonucleotides SD42mer/SD40mer (SEQ ID NO:8/SEQ ID NO:9)

   ClaI          SacI        XhoI        HpaI SD42mer5′ CGATTACTAGATCTGAGCTCCCCGGGCTCGAGGGATCCGTT  3′ SD40mer3′   TAATGATCTAGACTCGAGGGGCCCGAGCTCCCTAGGCAA  5′           BglII       SmaI        BamHIgenerating plasmid pSD478. Next the EcoRI site at the pUC/vacciniajunction was destroyed by digestion of pSD478 with EcoRI followed byblunt ending with Klenow fragment of E. coli polymerase and ligation,generating plasmid pSD478E⁻. pSD478E⁻ was digested with BamHI and HpaIand ligated with annealed synthetic oligonucleotides HEM5/HEM6 (SEQ IDNO:10/SEQ ID NO:11)

  BamHI EcoRI   HpaI HEM5 5′  GATCCGAATTCTAGCT 3′ HEM63′      GCTTAAGATCGA 5′generating plasmid pSD486. pSD486 was used as donor plasmid forrecombination with recombinant vaccinia virus vP533, generating vP553,which was isolated as a clear plaque in the presence of X-gal.

Example 3 Construction of Plasmid pMP494Δ for Deletion of ATI Region(A26L)

Referring now to FIG. 3, pSD414 contains SalI B cloned into pUC8. Toremove unwanted DNA sequences to the left of the A26L region, pSD414 wascut with XbaI within vaccinia sequences (pos. 137,079) and with HindIIIat the pUC/vaccinia junction, then blunt ended with Klenow fragment ofE. coli polymerase and ligated, resulting in plasmid pSD483. To removeunwanted vaccinia DNA sequences to the right of the A26L region, pSD483was cut with EcoRI (pos. 140,665 and at the pUC/vaccinia junction) andligated, forming plasmid pSD484. To remove the A26L coding region,pSD484 was cut with NdeI (partial) slightly upstream from the A26L ORF(pos. 139,004) and with HpaI (pos. 137,889) slightly downstream from theA26L ORF. The 5.2 kb vector fragment was isolated and ligated withannealed synthetic oligonucleotides ATI3/ATI4 (SEQ ID NO:12/SEQ IDNO:13)

  NdeI ATI3 5′ TATGAGTAACTTAACTCTTTTGTTAATTAAAAGTATATTCAA ATI43′   ACTCATTGAATTGAGAAAACAATTAATTTTCATATAAGTT                 BglII EcoRI HpaIAAAATAAGTTATATAAATAGATCTGAATTCGTT  3′ ATI3TTTTATTCAATATATTTATCTAGACTTAAGCAA  5′ ATI4reconstructing the region upstream from A26L and replacing the A26L ORFwith a short polylinker region containing the restriction sites BglII,EcoRI and HpaI, as indicated above. The resulting plasmid was designatedpSD485. Since the BglII and EcoRI sites in the polylinker region ofpSD485 are not unique, unwanted BglII and EcoRI sites were removed fromplasmid pSD483 (described above) by digestion with BglII (pos. 140,136)and with EcoRI at the pUC/vaccinia junction, followed by blunt endingwith Klenow fragment of E. coli polymerase and ligation. The resultingplasmid was designated pSD489. The 1.8 kb ClaI (pos. 137,198)/EcORV(pos. 139,048) fragment from pSD489 containing the A26L ORF was replacedwith the corresponding 0.7 kb polylinker-containing ClaI/EcoRV fragmentfrom pSD485, generating pSD492. The BglII and EcoRI sites in thepolylinker region of pSD492 are unique.

A 3.3 kb BglII cassette containing the E. coli Beta-galactosidase gene(Shapira et al., 1983) under the control of the vaccinia 11 kDa promoter(Bertholet et al., 1985; Perkus et al., 1990) was inserted into theBglII site of pSD492, forming pSD493KBG. Plasmid pSD493KBG was used inrecombination with rescuing virus vP553. Recombinant vaccinia virus,vP581, containing Beta-galactosidase in the A26L deletion region, wasisolated as a blue plaque in the presence of X-gal.

To generate a plasmid for the removal of Beta-galactosidase sequencesfrom vaccinia recombinant virus vP581, the polylinker region of plasmidpSD492 was deleted by mutagenesis (Mandecki, 1986) using syntheticoligonucleotide MPSYN177 (SEQ ID NO:14) (5′AAAATGGGCGTGGATTGTTAACTTTATATAACTTATTTTTTGAATATAC 3′). In the resultingplasmid, pMP494Δ, vaccinia DNA encompassing positions [137,889-138,937],including the entire A26L ORF is deleted. Recombination between thepMP494L and the Beta-galactosidase containing vaccinia recombinant,vP581, resulted in vaccinia deletion mutant vP618, which was isolated asa clear plaque in the presence of X-gal.

Example 4 Construction of Plasmid pSD467 for Deletion of HemagglutininGene (A56R)

Referring now to FIG. 4, vaccinia SalI G restriction fragment (pos. 160,744-173,351) crosses the HindIII A/B junction (pos. 162,539). pSD419contains vaccinia SalI G cloned into pUC8. The direction oftranscription for the hemagglutinin (HA) gene is indicated by an arrowin FIG. 4. Vaccinia sequences derived from HindIII B were removed bydigestion of pSD419 with HindIII within vaccinia sequences and at thepUC/vaccinia junction followed by ligation. The resulting plasmid,pSD456, contains the HA gene, A56R, flanked by 0.4 kb of vacciniasequences to the left and 0.4 kb of vaccinia sequences to the right.A56R coding sequences were removed by cutting pSD456 with RsaI (partial;pos. 161,090) upstream from A56R coding sequences, and with EagI (pos.162,054) near the end of the gene. The 3.6 kb RsaI/EagI vector fragmentfrom pSD456 was isolated and ligated with annealed syntheticoligonucleotides MPSYN59 (SEQ ID NO:15), MPSYN62 (SEQ ID NO:16), MPSYN60(SEQ ID NO:17), and MPSYN61 (SEQ ID NO:18)

  RsaI MPSYN59 5′ ACACGAATGATTTTCTAAAGTATTTGGAAAGTTTTATAGGT- MPSYN623′ TGTGCTTACTAAAAGATTTCATAAACCTTTCAAAATATCCA- MPSYN59  AGTTGATAGAACAAAATACATAATTT 3′ MPSYN62   TCAACTATCT 5′ MPSYN605′                 TGTAAAAATAAATCACTTTTTATA- MPSYN613′ TGTTTTATGTATTAAAACATTTTTATTTAGTGAAAAATAT-    BglII SmaI  PstI  EagIMPSYN60 CTAAGATCTCCCGGGCTGCAGC      3′ MPSYN61GATTCTAGAGGGCCCGACGTCGCCGG  5′reconstructing the DNA sequences upstream from the A56R ORF andreplacing the A56R ORF with a polylinker region as indicated above. Theresulting plasmid is pSD466. The vaccinia deletion in pSD466 encompassespositions [161,185-162,053]. The site of the deletion in pSD466 isindicated by a triangle in FIG. 4.

A 3.2 kb BglII/BamHI (partial) cassette containing the E. coliBeta-galactosidase gene (Shapira et al., 1983) under the control of thevaccinia 11 kDa promoter (Bertholet et al., 1985; Guo et al., 1989) wasinserted into the BglII site of pSD466, forming pSD466 KBG. PlasmidpSD466 KBG was used in recombination with rescuing virus vP618.Recombinant vaccinia virus, vP708, containing Beta-galactosidase in theA56R deletion, was isolated as a blue plaque in the presence of X-gal.

Beta-galactosidase sequences were deleted from vP708 using donor plasmidpSD467. pSD467 is identical to pSD466, except that EcoRI, SmaI and BamHIsites were removed from the pUC/vaccinia junction by digestion of pSD466with EcoRI/BamHI followed by blunt ending with Klenow fragment of E.coli polymerase and ligation. Recombination between vP708 and pSD467resulted in recombinant vaccinia deletion mutant, vP723, which wasisolated as a clear plaque in the presence of X-gal.

Example 5 Construction of Plasmid pMPCSK1Δ for Deletion of Open ReadingFrames [C7L-K1L]

Referring now to FIG. 5, the following vaccinia clones were utilized inthe construction of pMPCSK1Δ. pSD420 is SalI H cloned into pUC8. pSD435is KpnI F cloned into pUC18. pSD435 was cut with SphI and religated,forming pSD451. In pSD451, DNA sequences to the left of the SphI site(pos. 27,416) in HindIII M are removed (Perkus et al., 1990). pSD409 isHindIII M cloned into pUC8.

To provide a substrate for the deletion of the [C7L-K1L] gene clusterfrom vaccinia, E. coli Beta-galactosidase was first inserted into thevaccinia M2L deletion locus (Guo et al., 1990) as follows. To eliminatethe BglII site in pSD409, the plasmid was cut with BglII in vacciniasequences (pos. 28,212) and with BamHI at the pUC/vaccinia junction,then ligated to form plasmid pMP409B. pMP409B was cut at the unique SphIsite (pos. 27,416). M2L coding sequences were removed by mutagenesis(Guo et al., 1990; Mandecki, 1986) using synthetic oligonucleotide

MPSYN82 (SEQ ID NO: 19)                           BglII5′ TTTCTGTATATTTGCACCAATTTAGATCTT-ACTCAAAATATGTAAC AATA 3′The resulting plasmid, pMP409D, contains a unique BglII site insertedinto the M2L deletion locus as indicated above. A 3.2 kb BamHI(partial)/BglII cassette containing the E. coli Beta-galactosidase gene(Shapira et al., 1983) under the control of the 11 kDa promoter(Bertholet et al., 1985) was inserted into pMP409D cut with BglII. Theresulting plasmid, pMP409DBG (Guo et al., 1990), was used as donorplasmid for recombination with rescuing vaccinia virus vP723.Recombinant vaccinia virus, vP784, containing Beta-galactosidaseinserted into the M2L deletion locus, was isolated as a blue plaque inthe presence of X-gal.

A plasmid deleted for vaccinia genes [C7L-K1L] was assembled in pUC8 cutwith SmaI, HindIII and blunt ended with Klenow fragment of E. colipolymerase. The left flanking arm consisting of vaccinia HindIII Csequences was obtained by digestion of pSD420 with XbaI (pos. 18,628)followed by blunt ending with Klenow fragment of E. coli polymerase anddigestion with BglII (pos. 19,706). The right flanking arm consisting ofvaccinia HindIII K sequences was obtained by digestion of pSD451 withBglII (pos. 29,062) and EcoRV (pos. 29,778). The resulting plasmid,pMP581CK is deleted for vaccinia sequences between the BglII site (pos.19,706) in HindIII C and the BglII site (pos. 29,062) in HindIII K. Thesite of the deletion of vaccinia sequences in plasmid pMP581CK isindicated by a triangle in FIG. 5.

To remove excess DNA at the vaccinia deletion junction, plasmidpMP581CK, was cut at the NcoI sites within vaccinia sequences (pos.18,811; 19,655), treated with Bal-31 exonuclease and subjected tomutagenesis (Mandecki, 1986) using synthetic oligonucleotide MPSYN233(SEQ ID NO:20) 5′-TGTCATTTAACACTATACTCATATTAATAAAAATAATATTTATT-3′. Theresulting plasmid, pMPCSK18, is deleted for vaccinia sequences positions18,805-29,108, encompassing 12 vaccinia open reading frames [C7L-K1L].Recombination between pMPCSK1Δ and the Beta-galactosidase containingvaccinia recombinant, vP784, resulted in vaccinia deletion mutant,vP804, which was isolated as a clear plaque in the presence of X-gal.

Example 6 Construction of Plasmid pSD548 for Deletion of Large Subunit,Ribonucleotide Reductase (I4L)

Referring now to FIG. 6, plasmid pSD405 contains vaccinia HindIII I(pos. 63, 875-70,367) cloned in pUC8. pSD405 was digested with EcoRVwithin vaccinia sequences (pos. 67,933) and with SmaI at thepUC/vaccinia junction, and ligated, forming plasmid pSD518. pSD518 wasused as the source of all the vaccinia restriction fragments used in theconstruction of pSD548.

The vaccinia I4L gene extends from position 67,371-65,059. Direction oftranscription for I4L is indicated by an arrow in FIG. 6. To obtain avector plasmid fragment deleted for a portion of the I4L codingsequences, pSD518 was digested with BamHI (pos. 65,381) and HpaI (pos.67,001) and blunt ended using Klenow fragment of E. coli polymerase.This 4.8 kb vector fragment was ligated with a 3.2 kb SmaI cassettecontaining the E. coli Beta-galactosidase gene (Shapira et al., 1983)under the control of the vaccinia 11 kDa promoter (Bertholet et al.,1985; Perkus et al., 1990), resulting in plasmid pSD524 KBG. pSD524 KBGwas used as donor plasmid for recombination with vaccinia virus vP804.Recombinant vaccinia virus, vP855, containing Beta-galactosidase in apartial deletion of the I4L gene, was isolated as a blue plaque in thepresence of X-gal.

To delete Beta-galactosidase and the remainder of the I4L ORF fromvP855, deletion plasmid pSD548 was constructed. The left and rightvaccinia flanking arms were assembled separately in pUC8 as detailedbelow and presented schematically in FIG. 6.

To construct a vector plasmid to accept the left vaccinia flanking arm,pUC8 was cut with BamHI/EcoRI and ligated with annealed syntheticoligonucleotides 518A1/518A2

(SEQ ID NO: 21/SEQ ID NO: 22)    BamHI   RsaI 518A15′ GATCCTGAGTACTTTGTAATATAATGATATATATTTTCACT 518A23′     GACTCATGAAACATTATATTACTATATATAAAAGTGA                     BglII    EcoRITTATCTCATTTGAGAATAAAAAGATCTTAGG     3′ AATAGAGTAAACTCTTATTTTTCTAGAATCCTTAA 5′  518A1 518A2forming plasmid pSD531. pSD531 was cut with RsaI (partial) and BamHI anda 2.7 kb vector fragment isolated. pSD518 was cut with BglII (pos.64,459)/RsaI (pos. 64,994) and a 0.5 kb fragment isolated. The twofragments were ligated together, forming pSD537, which contains thecomplete vaccinia flanking arm left of the I4L coding sequences.

To construct a vector plasmid to accept the right vaccinia flanking arm,pUC8 was cut with BamHI/EcoRI and ligated with annealed syntheticoligonucleotides 518B1/518B2

(SEQ ID NO: 23/SEQ ID NO: 24)   BamHI BglII SmaI 518B15′ GATCCAGATCTCCCGGGAAAAAAATTATTTAACTTTTCAT 518B23′     GTCTAGAGGCCCCTTTTTTTAATAAATTGAAAAGTA                         RsaI   EcoRITAATAG-GGATTTGACGTATGTAGCGTACTAGG     3′ATTATC-CCTAAACTGCATACTACGCATGATCCTTAA 5′ 518B1 518B2forming plasmid pSD532. pSD532 was cut with RsaI (partial)/EcoRI and a2.7 kb vector fragment isolated. pSD518 was cut with RsaI withinvaccinia sequences (pos. 67,436) and EcoRI at the vaccinia/pUC junction,and a 0.6 kb fragment isolated. The two fragments were ligated together,forming pSD538, which contains the complete vaccinia flanking arm to theright of I4L coding sequences.

The right vaccinia flanking arm was isolated as a 0.6 kb EcoRI/BglIIfragment from pSD538 and ligated into pSD537 vector plasmid cut withEcoRI/BglII. In the resulting plasmid, pSD539, the I4L ORF (pos. 65,047-67,386) is replaced by a polylinker region, which is flanked by 0.6kb vaccinia DNA to the left and 0.6 kb vaccinia DNA to the right, all ina pUC background. The site of deletion within vaccinia sequences isindicated by a triangle in FIG. 6. To avoid possible recombination ofBeta-galactosidase sequences in the pUC-derived portion of pSD539 withBeta-galactosidase sequences in recombinant vaccinia virus vP855, thevaccinia I4L deletion cassette was moved from pSD539 into pRC11, a pUCderivative from which all Beta-galactosidase sequences have been removedand replaced with a polylinker region (Colinas et al., 1990). pSD539 wascut with EcoRI/PstI and the 1.2 kb fragment isolated. This fragment wasligated into pRC11 cut with EcoRI/PstI (2.35 kb), forming pSD548.Recombination between pSD548 and the Beta-galactosidase containingvaccinia recombinant, vP855, resulted in vaccinia deletion mutant vP866,which was isolated as a clear plaque in the presence of X-gal.

DNA from recombinant vaccinia virus vP866 was analyzed by restrictiondigests followed by electrophoresis on an agarose gel. The restrictionpatterns were as expected. Polymerase chain reactions (PCR) (Engelke etal., 1988) using vP866 as template and primers flanking the six deletionloci detailed above produced DNA fragments of the expected sizes.Sequence analysis of the PCR generated fragments around the areas of thedeletion junctions confirmed that the junctions were as expected.Recombinant vaccinia virus vP866, containing the six engineereddeletions as described above, was designated vaccinia vaccine strain“NYVAC.”

NYVAC was deposited under the terms of the Budapest Treaty on Mar. 6,1997 with the American Type Culture Collection (ATCC), P.O. Box 1549,Manassas, Va. 20108 USA under ATCC accession number VR-2559.

Example 7 Insertion of a Rabies Glycoprotein G Gene into NYVAC

The gene encoding rabies glycoprotein G under the control of thevaccinia H6 promoter (Taylor et al., 1988a,b) was inserted into TKdeletion plasmid pSD513. pSD513 is identical to plasmid pSD460 (FIG. 1)except for the presence of a polylinker region.

Referring now to FIG. 7, the polylinker region was inserted by cuttingpSD460 with SmaI and ligating the plasmid vector with annealed syntheticoligonucleotides VQ1A/VQ1B (SEQ ID NO:25/SEQ ID NO:26)

  SmaI BglII XhoI  PstI  NarI  BamHI VQ1A5′  GGGAGATCTCTCGAGCTGCAGGGCGCCGGATCCTTTTTCT 3′ VQ1B3′  CCCTCTAGAGAGCTCGACGTCCCGCGGCCTAGGAAAAAGA 5′to form vector plasmid pSD513. pSD513 was cut with SmaI and ligated witha SmaI ended 1.8 kb cassette containing the gene encoding the rabiesglycoprotein G gene under the control of the vaccinia H6 promoter(Taylor et al., 1988a,b). The resulting plasmid was designated pRW842.pRW842 was used as donor plasmid for recombination with NYVAC rescuingvirus (vP866). Recombinant vaccinia virus vP879 was identified by plaquehybridization using ³²P-labelled DNA probe to rabies glycoprotein Gcoding sequences.

The modified recombinant viruses of the present invention provideadvantages as recombinant vaccine vectors. The attenuated virulence ofthe vector advantageously reduces the opportunity for the possibility ofa runaway infection due to vaccination in the vaccinated individual andalso diminishes transmission from vaccinated to unvaccinated individualsor contamination of the environment.

The modified recombinant viruses are also advantageously used in amethod for expressing a gene product in a cell cultured in vitro byintroducing into the cell the modified recombinant virus having foreignDNA which codes for and expresses gene products in the cell.

Example 8 Construction of TROVAC-NDV Expressing the Fusion andHemagglutinin-Neuraminidase Glycoproteins of Newcastle Disease Virus

This example describes the development of TROVAC, a fowlpox virus vectorand, of a fowlpox Newcastle Disease Virus recombinant designatedTROVAC-NDV and its safety and efficacy. A fowlpox virus (FPV) vectorexpressing both F and HN genes of the virulent NDV strain Texas wasconstructed. The recombinant produced was designated TROVAC-NDV.TROVAC-NDV expresses authentically processed NDV glycoproteins in aviancells infected with the recombinant virus and inoculation of day oldchicks protects against subsequent virulent NDV challenge.

Cells and Viruses. The Texas strain of NDV is a velogenic strain.Preparation of cDNA clones of the F and HN genes has been previouslydescribed (Taylor et al., 1990; Edbauer et al., 1990). The strain of FPVdesignated FP-1 has been described previously (Taylor et al., 1988a). Itis a vaccine strain useful in vaccination of day old chickens. Theparental virus strain Duvette was obtained in France as a fowlpox scabfrom a chicken. The virus was attenuated by approximately 50 serialpassages in chicken embryonated eggs followed by 25 passages on chickenembryo fibroblast cells. The virus was subjected to four successiveplaque purifications. One plaque isolate was further amplified inprimary CEF cells and a stock virus, designated as TROVAC, established.The stock virus used in the in vitro recombination test to produceTROVAC-NDV had been subjected to twelve passages in primary CEF cellsfrom the plaque isolate.

TROVAC was deposited under the terms of the Budapest Treaty on Feb. 6,1997 with the American Type Culture Collection (ATCC), P.O. Box 1549,Manassas, Va. 20108 USA under ATCC accession number VR-2553.

Construction of a Cassette for NDV-F. A 1.8 kbp BamHI fragmentcontaining all but 22 nucleotides from the 5′ end of the F proteincoding sequence was excised from pNDV81 (Taylor et al., 1990) andinserted at the BamHI site of pUC18 to form pCE13. The vaccinia virus H6promoter previously described (Taylor et al., 1988a,b; Guo et al., 1989;Perkus et al., 1989) was inserted into pCE13 by digesting pCE13 withSalI, filling in the sticky ends with Klenow fragment of E. coli DNApolymerase and digesting with HindIII. A HindIII-EcoRV fragmentcontaining the H6 promoter sequence was then inserted into pCE13 to formpCE38. A perfect 5′ end was generated by digesting pCE38 with KpnI andNruI and inserting the annealed and kinased oligonucleotides CE75 (SEQID NO:27) and CE76 (SEQ ID NO:28) to generate pCE47.

CE75: CGATATCCGTTAAGTTTGTATCGTAATGGGCTCCAGATCTTCTACCAGGA TCCCGGTAC CE76:CGGGATCCTGGTAGAAGATCTGGAGCCCATTACGATACAAACTTAACGGA TATCG.In order to remove non-coding sequence from the 3′ end of the NDV-F aSmaI to PstI fragment from pCE13 was inserted into the SmaI and PstIsites of pUC18 to form pCE23. The non-coding sequences were removed bysequential digestion of pCE23 with SacI, BamHI, Exonuclease III, SInuclease and EcoRI. The annealed and kinased oligonucleotides CE42 (SEQID NO:29) and CE43 (SEQ ID NO:30) were then inserted to form pCE29.

CE42: AATTCGAGCTCCCCGGG CE43: CCCGGGGAGCTCGThe 3′ end of the NDV-F sequence was then inserted into plasmid pCE20already containing the 5′ end of NDV-F by cloning a PstI-SacI fragmentfrom pCE29 into the PstI and SacI sites of pCE20 to form pCE32.Generation of pCE20 has previously been described in Taylor et al.,1990.

In order to align the H6 promoter and NDV-F 5′ sequences contained inpCE47 with the 3′ NDV-F sequences contained in pCE32, a HindIII-PstIfragment of pCE47 was inserted into the HindIII and PstI sites of pCE32to form pCE49. The H6 promoted NDV-F sequences were then transferred tothe de-ORFed F8 locus (described below) by cloning a HindIII-NruIfragment from pCE49 into the HindIII and SmaI sites of pJCA002(described below) to form pCE54. Transcription stop signals wereinserted into pCE54 by digesting pCE54 with SacI, partially digestingwith BamHI and inserting the annealed and kinased oligonucleotides CE166(SEQ ID NO:31) and CE167 (SEQ ID NO:32) to generate pCE58.

CE166: CTTTTTATAAAAAGTTAACTACGTAG CE167:GATCCTACGTAGTTAACTTTTTATAAAAAGAGCTA perfect 3′ end for NDV-F was obtained by using the polymerase chainreaction (PCR) with pCE54 as template and oligonucleotides CE182 (SEQ IDNO:33) and CE183 (SEQ ID NO:34) as primers.

CE182: CTTAACTCAGCTGACTATCC CE183:TACGTAGTTAACTTTTTATAAAAATCATATTTTTGTAGTGGCTC

The PCR fragment was digested with PvuII and HpaI and cloned into pCE58that had been digested with HpaI and partially digested with PvuII. Theresulting plasmid was designated pCE64. Translation stop signals wereinserted by cloning a HindIII-HpaI fragment which contains the completeH6 promoter and F coding sequence from pCE64 into the HindIII and HpaIsites of pRW846 to generate pCE71, the final cassette for NDV-F. PlasmidpRW846 is essentially equivalent to plasmid pJCA002 (described below)but containing the H6 promoter and transcription and translation stopsignals. Digestion of pRW846 with HindIII and HpaI eliminates the H6promoter but leaves the stop signals intact.

Construction of Cassette for NDV-HN. Construction of plasmid pRW802 waspreviously described in Edbauer et al., 1990. This plasmid contains theNDV-HN sequences linked to the 3′ end of the vaccinia virus H6 promoterin a pUC9 vector. A HindIII-EcoRV fragment encompassing the 5′ end ofthe vaccinia virus H6 promoter was inserted into the HindIII and EcoRVsites of pRW802 to form pRW830. A perfect 3′ end for NDV-HN was obtainedby inserting the annealed and kinased oligonucleotides CE162 (SEQ IDNO:35) and CE163 (SEQ ID NO:36) into the EcoRI site of pRW830 to formpCE59, the final cassette for NDV-HN.

CE162: AATTCAGGATCGTTCCTTTACTAGTTGAGATTCTCAAGGATGATGGGATTTAATTTTTATAAGCTTG CE163:AATTCAAGCTTATAAAAATTAAATCCCATCATCCTTGAGAATCTCAACTA GTAAAGGAACGATCCTG

Construction of FPV Insertion Vector. Plasmid pRW731-15 contains a 10 kbPvuII-PvuII fragment cloned from genomic DNA. The nucleotide sequencewas determined on both strands for a 3660 bp PvuII-EcoRV fragment and isshown in FIG. 11 (SEQ ID NO:67). The limits of an open reading framedesignated here as F8 were determined. Plasmid pRW761 is a sub-clone ofpRW731-15 containing a 2430 bp EcoRV-EcoRV fragment. The F8 ORF wasentirely contained between an XbaI site and an SspI site in pRW761. Inorder to create an insertion plasmid which on recombination with TROVACgenomic DNA would eliminate the F8 ORF, the following steps werefollowed. Plasmid pRW761 was completely digested with XbaI and partiallydigested with SspI. A 3700 bp XbaI-SspI band was isolated from the geland ligated with the annealed double-stranded oligonucleotides JCA017(SEQ ID NO:37) and JCA018 (SEQ ID NO:38).

JCA017: 5′ CTAGACACTTTATGTTTTTTAATATCCGGTCTTAAAAGCTTCCCGGGGATCCTTATACGGGGAATAAT JCA018:5′ ATTATTCCCCGTATAAGGATCCCCCGGGAAGCTTTTAAGACCGGATA TTAAAAAACATAAAGTGTThe plasmid resulting from this ligation was designated pJCA002.

Construction of Double Insertion Vector for NDV F and HN. The H6promoted NDV-HN sequence was inserted into the H6 promoted NDV-Fcassette by cloning a HindIII fragment from pCE59 that had been filledin with Klenow fragment of E. coli DNA polymerase into the HpaI site ofpCE71 to form pCE80. Plasmid pCE80 was completely digested with NdeI andpartially digested with BglII to generate an NdeI-BglII 4760 bp fragmentcontaining the NDV F and HN genes both driven by the H6 promoter andlinked to F8 flanking arms. Plasmid pJCA021 was obtained by inserting a4900 bp PvuII-HindIII fragment from pRW731-15 into the SmaI and HindIIIsites of pBSSK+. Plasmid pJCA021 was then digested with NdeI and BglIIand ligated to the 4760 bp NdeI-BglII fragment of pCE80 to form pJCA024.Plasmid pJCA024 therefore contains the NDV-F and HN genes inserted inopposite orientation with 3′ ends adjacent between FPV flanking arms.Both genes are linked to the vaccinia virus H6 promoter. The rightflanking arm adjacent to the NDV-F sequence consists of 2350 bp of FPVsequence. The left flanking arm adjacent to the NDV-HN sequence consistsof 1700 bp of FPV sequence.

Development of TROVAC-NDV. Plasmid pJCA024 was transfected into TROVACinfected primary CEF cells by using the calcium phosphate precipitationmethod previously described (Panicali et al., 1982; Piccini et al.,1987). Positive plaques were selected on the basis of hybridization tospecific NDV-F and HN radiolabelled probes and subjected to fivesequential rounds of plaque purification until a pure population wasachieved. One representative plaque was then amplified and the resultingTROVAC recombinant was designated TROVAC-NDV (vFP96).

Immunofluorescence. Indirect immunofluorescence was performed asdescribed (Taylor et al., 1990) using a polyclonal anti-NDV serum and,as mono-specific reagents, sera produced in rabbits against vacciniavirus recombinants expressing NDV-F or NDV-HN.

Immunoprecipitation. Immunoprecipitation reactions were performed asdescribed (Taylor et al., 1990) using a polyclonal anti-NDV serumobtained from SPAFAS Inc., Storrs, Conn.

The stock virus was screened by in situ plaque hybridization to confirmthat the F8 ORF was deleted. The correct insertion of the NDV genes intothe TROVAC genome and the deletion of the F8 ORF was also confirmed bySouthern blot hybridization.

In NDV-infected cells, the F glycoprotein is anchored in the membranevia a hydrophobic transmembrane region near the carboxyl terminus andrequires post-translational cleavage of a precursor, F₀, into twodisulfide linked polypeptides F₁ and F₂. Cleavage of F₀ is important indetermining the pathogenicity of a given NDV strain (Homma and Ohuchi,1973; Nagai et al., 1976; Nagai et al., 1980), and the sequence of aminoacids at the cleavage site is therefore critical in determining viralvirulence. It has been determined that amino acids at the cleavage sitein the NDV-F sequence inserted into FPV to form recombinant vFP29 hadthe sequence Arg-Arg-Gln-Arg-Arg (SEQ ID NO: 149) (Taylor et al., 1990)which conforms to the sequence found to be a requirement for virulentNDV strains (Chambers et al., 1986; Espion et al., 1987; Le et al.,1988; McGinnes and Morrison, 1986; Toyoda et al., 1987). The HNglycoprotein synthesized in cells infected with virulent strains of NDVis an uncleaved glycoprotein of 74 kDa. Extremely avirulent strains suchas Ulster and Queensland encode an HN precursor (HNo) which requirescleavage for activation (Garten et al., 1980).

The expression of F and HN genes in TROVAC-NDV was analyzed to confirmthat the gene products were authentically processed and presented.Indirect-immunofluorescence using a polyclonal anti-NDV chicken serumconfirmed that immunoreactive proteins were presented on the infectedcell surface. To determine that both proteins were presented on theplasma membrane, mono-specific rabbit sera were produced againstvaccinia recombinants expressing either the F or HN glycoproteins.Indirect immunofluorescence using these sera confirmed the surfacepresentation of both proteins.

Immunoprecipitation experiments were performed by using (³⁵S) methioninelabeled lysates of CEF cells infected with parental and recombinantviruses. The expected values of apparent molecular weights of theglycosylated forms of F₁ and F₂ are 54.7 and 10.3 kDa respectively(Chambers et al., 1986). In the immunoprecipitation experiments using apolyclonal anti-NDV serum, fusion specific products of the appropriatesize were detected from the NDV-F single recombinant vFP29 (Taylor etal., 1990) and the TROVAC-NDV double recombinant vFP96. The HNglycoprotein of appropriate size was also detected from the NDV-HNsingle recombinant VFP-47 (Edbauer et al., 1990) and TROVAC-NDV. No NDVspecific products were detected from uninfected and parental TROVACinfected CEF cells.

In CEF cells, the F and HN glycoproteins are appropriately presented onthe infected cell surface where they are recognized by NDV immune serum.Immunoprecipitation analysis indicated that the F₀ protein isauthentically cleaved to the F₁ and F2 components required in virulentstrains. Similarly, the HN glycoprotein was authentically processed inCEF cells infected with recombinant TROVAC-NDV.

Previous reports (Taylor et al., 1990; Edbauer et al., 1990; Boursnellet al., 1990a,b,c; Ogawa et al., 1990) would indicate that expression ofeither HN or F alone is sufficient to elicit protective immunity againstNDV challenge. Work on other paramyxoviruses has indicated, however,that antibody to both proteins may be required for full protectiveimmunity. It has been demonstrated that SV5 virus could spread in tissueculture in the presence of antibody to the HN glycoprotein but not tothe F glycoprotein (Merz et al., 1980). In addition, it has beensuggested that vaccine failures with killed measles virus vaccines weredue to inactivation of the fusion component (Norrby et al., 1975). Sinceboth NDV glycoproteins have been shown to be responsible for elicitingvirus neutralizing antibody (Avery et al., 1979) and both glycoproteins,when expressed individually in a fowlpox vector are able to induce aprotective immune response, it can be appreciated that the mostefficacious NDV vaccine should express both glycoproteins.

Example 9 Construction of ALVAC Recombinants Expressing Rabies VirusGlycoprotein G

This example describes the development of ALVAC, a canarypox virusvector and, of a canarypox-rabies recombinant designated as ALVAC-RG(vCP65) and its safety and efficacy.

Cells and Viruses. The parental canarypox virus (Rentschler strain) is avaccinal strain for canaries. The vaccine strain was obtained from awild type isolate and attenuated through more than 200 serial passageson chick embryo fibroblasts. A master viral seed was subjected to foursuccessive plaque purifications under agar and one plaque clone wasamplified through five additional passages after which the stock viruswas used as the parental virus in in vitro recombination tests. Theplaque purified canarypox isolate is designated ALVAC.

ALVAC was deposited under the terms of the Budapest Treaty with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md., 20852, USA: ALVAC under ATCC accession number VR-2547 onNov. 14, 1996.

Construction of a Canarypox Insertion Vector. An 880 bp canarypox PvuIIfragment was cloned between the PvuII sites of pUC9 to form pRW764.5.The sequence of this fragment is shown in FIG. 8 (SEQ ID NO:66) betweenpositions 1372 and 2251. The limits of an open reading frame designatedas C5 were defined. It was determined that the open reading frame wasinitiated at position 166 within the fragment and terminated at position487. The C5 deletion was made without interruption of open readingframes. Bases from position 167 through position 455 were replaced withthe sequence (SEQ ID NO:39) GCTTCCCGGGAATTCTAGCTAGCTAGTTT. Thisreplacement sequence contains HindIII, SmaI and EcoRI insertion sitesfollowed by translation stops and a transcription termination signalrecognized by vaccinia virus RNA polymerase (Yuen et al., 1987).Deletion of the C5 ORF was performed as described below. PlasmidpRW764.5 was partially cut with RsaI and the linear product wasisolated. The RsaI linear fragment was recut with BglII and the pRW764.5fragment now with a RsaI to BglII deletion from position 156 to position462 was isolated and used as a vector for the following syntheticoligonucleotides:

RW145 (SEQ ID NO: 40): ACTCTCAAAAGCTTCCCGGGAATTCTAGCTAGCTAGTTTTTATAAARW146 (SEQ ID NO: 41):GATCTTTATAAAAACTAGCTAGCTAGAATTCCCGGGAAGCTTTTGAGAGTOligonucleotides RW145 and RW146 were annealed and inserted into the pRW764.5 RsaI and BglII vector described above. The resulting plasmid isdesignated pRW831.

Construction of Insertion Vector Containing the Rabies G Gene.Construction of pRW838 is illustrated below. Oligonucleotides A throughE, which overlap the translation initiation codon of the H6 promoterwith the ATG of rabies G, were cloned into pUC9 as pRW737.Oligonucleotides A through E contain the H6 promoter, starting at NruI,through the HindIII site of rabies G followed by BglII. Sequences ofoligonucleotides A through E ((SEQ ID NO:42)-(SEQ ID NO:46)) are:

A (SEQ ID NO: 42): CTGAAATTATTTCATTATCGCGATATCCGTTAAGTTTGTATCGTAATGGTTCCTCAGGCTCTCCTGTTTGT B (SEQ ID NO: 43):CATTACGATACAAACTTAACGGATATCGCGATAATGAAATAATTTCAG C (SEQ ID NO: 44):ACCCCTTCTGGTTTTTCCGTTGTGTTTTGGGAAATTCCCTATTTACACGATCCCAGACAAGCTTAGATCTCAG D (SEQ ID NO: 45):CTGAGATCTAAGCTTGTCTGGGATCGTGTAAATAGGGAATTTCCCAAAAC A E (SEQ ID NO: 46):CAACGGAAAAACCAGAAGGGGTACAAACAGGAGAGCCTGAGGAACThe diagram of annealed oligonucleotides A through E is as follows:

Oligonucleotides A through E were kinased, annealed (95° C. for 5minutes, then cooled to room temperature), and inserted between thePvuII sites of pUC9. The resulting plasmid, pRW737, was cut with HindIIIand BglII and used as a vector for the 1.6 kbp HindIII-BglII fragment ofptg155PRO (Kieny et al., 1984) generating pRW739. The ptg155PRO HindIIIsite is 86 bp downstream of the rabies G translation initiation codon.BglII is downstream of the rabies G translation stop codon in ptg155PRO.pRW739 was partially cut with NruI, completely cut with BglII, and a 1.7kbp NruI-BglII fragment, containing the 3′ end of the H6 promoterpreviously described (Taylor et al., 1988a,b; Guo et al., 1989; Perkuset al., 1989) through the entire rabies G gene, was inserted between theNruI and BamHI sites of pRW824. The resulting plasmid is designatedpRW832. Insertion into pRW824 added the H6 promoter 5′ of NruI. ThepRW824 sequence of BamHI followed by SmaI is (SEQ ID NO:47):GGATCCCCGGG. pRW824 is a plasmid that contains a nonpertinent genelinked precisely to the vaccinia virus H6 promoter. Digestion with NruIand BamHI completely excised this nonpertinent gene. The 1.8 kbp pRW832SmaI fragment, containing H6 promoted rabies G, was inserted into theSmaI of pRW831, to form plasmid pRW838.

Development of ALVAC-RG. Plasmid pRW838 was transfected into ALVACinfected primary CEF cells by using the calcium phosphate precipitationmethod previously described (Panicali et al., 1982; Piccini et al.,1987). Positive plaques were selected on the basis of hybridization to aspecific rabies G probe and subjected to 6 sequential rounds of plaquepurification until a pure population was achieved. One representativeplaque was then amplified and the resulting ALVAC recombinant wasdesignated ALVAC-RG (vCP65) (see also FIGS. 9A and 9B). The correctinsertion of the rabies G gene into the ALVAC genome without subsequentmutation was confirmed by sequence analysis.

Immunofluorescence. During the final stages of assembly of mature rabiesvirus particles, the glycoprotein component is transported from thegolgi apparatus to the plasma membrane where it accumulates with thecarboxy terminus extending into the cytoplasm and the bulk of theprotein on the external surface of the cell membrane. In order toconfirm that the rabies glycoprotein expressed in ALVAC-RG was correctlypresented, immunofluorescence was performed on primary CEF cellsinfected with ALVAC or ALVAC-RG. Immunofluorescence was performed aspreviously described (Taylor et al., 1990) using a rabies G monoclonalantibody. Strong surface fluorescence was detected on CEF cells infectedwith ALVAC-RG but not with the parental ALVAC.

Immunoprecipitation. Preformed monolayers of primary CEF, Vero (a lineof African Green monkey kidney cells ATCC # CCL81) and MRC-5 cells (afibroblast-like cell line derived from normal human fetal lung tissueATCC # CCL171) were inoculated at 10 pfu per cell with parental virusALVAC and recombinant virus ALVAC-RG in the presence of radiolabelled³⁵S-methionine and treated as previously described (Taylor et al.,1990). Immunoprecipitation reactions were performed using a rabies Gspecific monoclonal antibody. Efficient expression of a rabies specificglycoprotein with a molecular weight of approximately 67 kDa wasdetected with the recombinant ALVAC-RG. No rabies specific products weredetected in uninfected cells or cells infected with the parental ALVACvirus.

Sequential Passaging Experiment. In studies with ALVAC virus in a rangeof non-avian species no proliferative infection or overt disease wasobserved (Taylor et al., 1991b). However, in order to establish thatneither the parental nor recombinant virus could be adapted to grow innon-avian cells, a sequential passaging experiment was performed.

The two viruses, ALVAC and ALVAC-RG, were inoculated in 10 sequentialblind passages in three cell substrates:

-   -   (1) Primary chick embryo fibroblast (CEF) cells produced from 11        day old white leghorn embryos;    -   (2) Vero cells—a continuous line of African Green monkey kidney        cells (ATCC # CCL81); and    -   (3) MRC-5 cells—a diploid cell line derived from human fetal        lung tissue (ATCC # CCL171).        The initial inoculation was performed at an m.o.i. of 0.1 pfu        per cell using three 60 mm dishes of each cell substrate        containing 2×10⁶ cells per dish. One dish was inoculated in the        presence of 40 μg/ml of Cytosine arabinoside (Ara C), an        inhibitor of DNA replication. After an absorption period of 1        hour at 37° C., the inoculum was removed and the monolayer        washed to remove unabsorbed virus. At this time the medium was        replaced with 5 ml of EMEM+2% NBCS on two dishes (samples t0 and        t7) and 5 ml of EMEM+2% NBCS containing 40 μg/ml Ara C on the        third (sample t7A). Sample t0 was frozen at −70° C. to provide        an indication of the residual input virus. Samples t7 and t7A        were incubated at 37° C. for 7 days, after which time the        contents were harvested and the cells disrupted by indirect        sonication.

One ml of sample t7 of each cell substrate was inoculated undiluted ontothree dishes of the same cell substrate (to provide samples t0, t7 andt7A) and onto one dish of primary CEF cells. Samples to, t7 and t7A weretreated as for passage one. The additional inoculation on CEF cells wasincluded to provide an amplification step for more sensitive detectionof virus which might be present in the non-avian cells.

This procedure was repeated for 10 (CEF and MRC-5) or 8 (Vero)sequential blind passages. Samples were then frozen and thawed threetimes and assayed by titration on primary CEF monolayers.

Virus yield in each sample was then determined by plaque titration onCEF monolayers under agarose. Summarized results of the experiment areshown in Tables 1 and 2.

The results indicate that both the parental ALVAC and the recombinantALVAC-RG are capable of sustained replication on CEF monolayers with noloss of titer. In Vero cells, levels of virus fell below the level ofdetection after 2 passages for ALVAC and 1 passage for ALVAC-RG. InMRC-5 cells, a similar result was evident, and no virus was detectedafter 1 passage. Although the results for only four passages are shownin Tables 1 and 2 the series was continued for 8 (Vero) and 10 (MRC-5)passages with no detectable adaptation of either virus to growth in thenon-avian cells.

In passage 1 relatively high levels of virus were present in the t7sample in MRC-5 and Vero cells. However this level of virus wasequivalent to that seen in the t0 sample and the t7A sample incubated inthe presence of Cytosine arabinoside in which no viral replication canoccur. This demonstrated that the levels of virus seen at 7 days innon-avian cells represented residual virus and not newly replicatedvirus.

In order to make the assay more sensitive, a portion of the 7 dayharvest from each cell substrate was inoculated onto a permissive CEFmonolayer and harvested at cytopathic effect (CPE) or at 7 days if noCPE was evident. The results of this experiment are shown in Table 3.Even after amplification through a permissive cell substrate, virus wasonly detected in MRC-5 and Vero cells for two additional passages. Theseresults indicated that under the conditions used, there was noadaptation of either virus to growth in Vero or MRC-5 cells.

Inoculation of Macaques. Four HIV seropositive macaques were initiallyinoculated with ALVAC-RG as described in Table 4. After 100 days theseanimals were re-inoculated to determine a booster effect, and anadditional seven animals were inoculated with a range of doses. Bloodwas drawn at appropriate intervals and sera analyzed, after heatinactivation at 56° C. for 30 minutes, for the presence of anti-rabiesantibody using the Rapid Fluorescent Focus Inhibition Assay (Smith etal., 1973).

Inoculation of Chimpanzees. Two adult male chimpanzees (50 to 65 kgweight range) were inoculated intramuscularly or subcutaneously with1×10⁷ pfu of vCP65. Animals were monitored for reactions and bled atregular intervals for analysis for the presence of anti-rabies antibodywith the RFFI test (Smith et al., 1973). Animals were re-inoculated withan equivalent dose 13 weeks after the initial inoculation.

Inoculation of Mice. Groups of mice were inoculated with 50 to 100 μl ofa range of dilutions of different batches of vCP65. Mice were inoculatedin the footpad. On day 14, mice were challenged by intracranialinoculation of from 15 to 43 mouse LD₅₀ of the virulent CVS strain ofrabies virus. Survival of mice was monitored and a protective dose 50%(PD₅₀) calculated at 28 days post-inoculation.

Inoculation of Dogs and Cats. Ten beagle dogs, 5 months old, and 10cats, 4 months old, were inoculated subcutaneously with either 6.7 or7.7 log₁₀ TCID₅₀ of ALVAC-RG. Four dogs and four cats were notinoculated. Animals were bled at 14 and 28 days post-inoculation andanti-rabies antibody assessed in an RFFI test. The animals receiving 6.7log₁₀ TCID₅₀ of ALVAC-RG were challenged at 29 days post-vaccinationwith 3.7 log₁₀ mouse LD₅₀ (dogs) or 4.3 log₁₀ mouse LD₅₀ (cats) of theNYGS rabies virus challenge strain.

Inoculation of Squirrel Monkeys. Three groups of four squirrel monkeys(Saimiri sciureus) were inoculated with one of three viruses (a) ALVAC,the parental canarypox virus, (b) ALVAC-RG, the recombinant expressingthe rabies G glycoprotein or (c) vCP37, a canarypox recombinantexpressing the envelope glycoprotein of feline leukemia virus.Inoculations were performed under ketamine anaesthesia. Each animalreceived at the same time: (1) 20 μl instilled on the surface of theright eye without scarification; (2) 100 μl as several droplets in themouth; (3) 100 μl in each of two intradermal injection sites in theshaven skin of the external face of the right arm; and (4) 100 μl in theanterior muscle of the right thigh.

Four monkeys were inoculated with each virus, two with a total of 5.0log₁₀ pfu and two with a total of 7.0 log₁₀ pfu. Animals were bled atregular intervals and sera analyzed for the presence of antirabiesantibody using an RFFI test (Smith et al., 1973). Animals were monitoreddaily for reactions to vaccination. Six months after the initialinoculation the four monkeys receiving ALVAC-RG, two monkeys initiallyreceiving vCP37, and two monkeys initially receiving ALVAC, as well asone naive monkey were inoculated with 6.5 log₁₀ pfu of ALVAC-RGsubcutaneously. Sera were monitored for the presence of rabiesneutralizing antibody in an RFFI test (Smith et al., 1973).

Inoculation of Human Cell Lines with ALVAC-RG. In order to determinewhether efficient expression of a foreign gene could be obtained innon-avian cells in which the virus does not productively replicate, fivecell types, one avian and four non-avian, were analyzed for virus yield,expression of the foreign rabies G gene and viral specific DNAaccumulation. The cells inoculated were:

-   -   (a) Vero, African Green monkey kidney cells, ATCC # CCL81;    -   (b) MRC-5, human embryonic lung, ATCC # CCL 171;    -   (c) WISH human amnion, ATCC # CCL 25;    -   (d) Detroit-532, human foreskin, Downs's syndrome, ATCC # CCL        54; and    -   (e) Primary CEF cells.

Chicken embryo fibroblast cells produced from 11 day old white leghornembryos were included as a positive control. All inoculations wereperformed on preformed monolayers of 2×10⁶ cells as discussed below.

A. Methods for DNA Analysis.

-   -   Three dishes of each cell line were inoculated at 5 pfu/cell of        the virus under test, allowing one extra dish of each cell line        un-inoculated. One dish was incubated in the presence of 40        μg/ml of cytosine arabinoside (Ara C). After an adsorption        period of 60 minutes at 37° C., the inoculum was removed and the        monolayer washed twice to remove unadsorbed virus. Medium (with        or without Ara C) was then replaced. Cells from one dish        (without Ara C) were harvested as a time zero sample. The        remaining dishes were incubated at 37° C. for 72 hours, at which        time the cells were harvested and used to analyze DNA        accumulation. Each sample of 2×10⁶ cells was resuspended in 0.5        ml phosphate buffered saline (PBS) containing 40 mM EDTA and        incubated for 5 minutes at 37° C. An equal volume of 1.5%        agarose prewarmed at 42° C. and containing 120 mM EDTA was added        to the cell suspension and gently mixed. The suspension was        transferred to an agarose plug mold and allowed to harden for at        least 15 min. The agarose plugs were then removed and incubated        for 12-16 hours at 50° C. in a volume of lysis buffer (1%        sarkosyl, 100 μg/ml proteinase K, 10 mM Tris HCl pH 7.5, 200 mM        EDTA) that completely covers the plug. The lysis buffer was then        replaced with 5.0 ml sterile 0.5×TBE (44.5 mM Tris-borate, 44.5        mM boric acid, 0.5 mM EDTA) and equilibrated at 4° C. for 6        hours with 3 changes of TBE buffer. The viral DNA within the        plug was fractionated from cellular RNA and DNA using a pulse        field electrophoresis system. Electrophoresis was performed for        20 hours at 180 V with a ramp of 50-90 sec at 15° C. in 0.5×TBE.        The DNA was run with lambda DNA molecular weight standards.        After electrophoresis the viral DNA band was visualized by        staining with ethidium bromide. The DNA was then transferred to        a nitrocellulose membrane and probed with a radiolabelled probe        prepared from purified ALVAC genomic DNA.

B. Estimation of Virus Yield.

-   -   Dishes were inoculated exactly as described above, with the        exception that input multiplicity was 0.1 pfu/cell. At 72 hours        post infection, cells were lysed by three successive cycles of        freezing and thawing. Virus yield was assessed by plaque        titration on CEF monolayers.

C. Analysis of Expression of Rabies G Gene.

-   -   Dishes were inoculated with recombinant or parental virus at a        multiplicity of 10 pfu/cell, allowing an additional dish as an        uninfected virus control. After a one hour absorption period,        the medium was removed and replaced with methionine free medium.        After a 30 minute period, this medium was replaced with        methionine-free medium containing 25 uCi/ml of ³⁵S-Methionine.        Infected cells were labelled overnight (approximately 16 hours),        then lysed by the addition of buffer A lysis buffer.        Immunoprecipitation was performed as previously described        (Taylor et al., 1990) using a rabies G specific monoclonal        antibody.

Results: Estimation of Viral Yield. The results of titration for yieldat 72 hours after inoculation at. 0.1 pfu per cell are shown in Table 5.The results indicate that while a productive infection can be attainedin the avian cells, no increase in virus yield can be detected by thismethod in the four non-avian cell systems.

Analysis of Viral DNA Accumulation. In order to determine whether theblock to productive viral replication in the non-avian cells occurredbefore or after DNA replication, DNA from the cell lysates wasfractionated by electrophoresis, transferred to nitrocellulose andprobed for the presence of viral specific DNA. DNA from uninfected CEFcells, ALVAC-RG infected CEF cells at time zero, ALVAC-RG infected CEFcells at 72 hours post-infection and ALVAC-RG infected CEF cells at 72hours post-infection in the presence of 40 pg/ml of cytosine arabinosideall showed some background activity, probably due to contaminating CEFcellular DNA in the radiolabelled ALVAC DNA probe preparation. However,ALVAC-RG infected CEF cells at 72 hours post-infection exhibited astrong band in the region of approximately 350 kbp representingALVAC-specific viral DNA accumulation. No such band is detectable whenthe culture is incubated in the presence of the DNA synthesis inhibitor,cytosine arabinoside. Equivalent samples produced in Vero cells showed avery faint band at approximately 350 kbp in the ALVAC-RG infected Verocells at time zero. This level represented residual virus. The intensityof the band was amplified at 72 hours post-infection indicating thatsome level of viral specific DNA replication had occurred in Vero cellswhich had not resulted in an increase in viral progeny. Equivalentsamples produced in MRC-5 cells indicated that no viral specific DNAaccumulation was detected under these conditions in this cell line. Thisexperiment was then extended to include additional human cell lines,specifically WISH and Detroit-532 cells. ALVAC infected CEF cells servedas a positive control. No viral specific DNA accumulation was detectedin either WISH or Detroit cells inoculated with ALVAC-RG. It should benoted that the limits of detection of this method have not been fullyascertained and viral DNA accumulation may be occurring, but at a levelbelow the sensitivity of the method. Other experiments in which viralDNA replication was measured by ³H-thymidine incorporation support theresults obtained with Vero and MRC-5 cells.

Analysis of Rabies Gene Expression. To determine if any viral geneexpression, particularly that of the inserted foreign gene, wasoccurring in the human cell lines even in the absence of viral DNAreplication, immunoprecipitation experiments were performed on³⁵S-methionine labelled lysates of avian and non-avian cells infectedwith ALVAC and ALVAC-RG. The results of immunoprecipitation using arabies G specific monoclonal antibody illustrated specificimmunoprecipitation of a 67 kDa glycoprotein in CEF, Vero and MRC-5,WISH and Detroit cells infected with ALVAC-RG. No such specific rabiesgene products were detected in any of the uninfected and parentallyinfected cell lysates.

The results of this experiment indicated that in the human cell linesanalyzed, although the ALVAC-RG recombinant was able to initiate aninfection and express a foreign gene product under the transcriptionalcontrol of the H6 early/late vaccinia virus promoter, the replicationdid not proceed through DNA replication, nor was there any detectableviral progeny produced. In the Vero cells, although some level ofALVAC-RG specific DNA accumulation was observed, no viral progeny wasdetected by these methods. These results would indicate that in thehuman cell lines analyzed the block to viral replication occurs prior tothe onset of DNA replication, while in Vero cells, the block occursfollowing the onset of viral DNA replication.

In order to determine whether the rabies glycoprotein expressed inALVAC-RG was immunogenic, a number of animal species were tested byinoculation of the recombinant. The efficacy of current rabies vaccinesis evaluated in a mouse model system. A similar test was thereforeperformed using ALVAC-RG. Nine different preparations of virus(including one vaccine batch (J) produced after 10 serial tissue culturepassages of the seed virus) with infectious titers ranging from 6.7 to8.4 log₁₀ TCID₅₀ per ml were serially diluted and 50 to 100 μl ofdilutions inoculated into the footpad of four to six week old mice. Micewere challenged 14 days later by the intracranial route with 300 μl ofthe CVS strain of rabies virus containing from 15 to 43 mouse LD₅₀ asdetermined by lethality titration in a control group of mice. Potency,expressed as the PD₅₀ (Protective dose 50%), was calculated at 14 dayspost-challenge. The results of the experiment are shown in Table 6. Theresults indicated that ALVAC-RG was consistently able to protect miceagainst rabies virus challenge with a PD₅₀ value ranging from 3.33 to4.56 with a mean value of 3.73 (STD 0.48). As an extension of thisstudy, male mice were inoculated intracranially with 50 μl of viruscontaining 6.0 log₁₀ TCID₅₀ of ALVAC-RG or with an equivalent volume ofan uninfected cell suspension. Mice were sacrificed on days 1, 3 and 6post-inoculation and their brains removed, fixed and sectioned.Histopathological examination showed no evidence for neurovirulence ofALVAC-RG in mice.

In order to evaluate the safety and efficacy of ALVAC-RG for dogs andcats, a group of 14, 5 month old beagles and 14, 4 month old cats wereanalyzed. Four animals in each species were not vaccinated. Five animalsreceived 6.7 log₁₀ TCID₅₀ subcutaneously and five animals received 7.7log₁₀ TCID₅₀ by the same route. Animals were bled for analysis foranti-rabies antibody. Animals receiving no inoculation or 6.7 log₁₀TCID₅₀ of ALVAC-RG were challenged at 29 days post-vaccination with 3.7log₁₀ mouse LD₅₀ (dogs, in the temporal muscle) or 4.3 log₁₀ mouse LD₅₀(cats, in the neck) of the NYGS rabies virus challenge strain. Theresults of the experiment are shown in Table 7.

No adverse reactions to inoculation were seen in either cats or dogswith either dose of inoculum virus. Four of 5 dogs immunized with 6.7log₁₀ TCID₅₀ had antibody titers on day 14 post-vaccination and all dogshad titers at 29 days. All dogs were protected from a challenge whichkilled three out of four controls. In cats, three of five cats receiving6.7 log₁₀ TCID₅₀ had specific antibody titers on day 14 and all catswere positive on day 29 although the mean antibody titer was low at 2.9IU. Three of five cats survived a challenge which killed all controls.All cats immunized with 7.7 log₁₀ TCID₅₀ had antibody titers on day 14and at day 29 the Geometric Mean Titer was calculated as 8.1International Units.

The immune response of squirrel monkeys (Saimiri sciureus) toinoculation with ALVAC, ALVAC-RG and an unrelated canarypox virusrecombinant was examined. Groups of monkeys were inoculated as describedabove and sera analyzed for the presence of rabies specific antibody.Apart from minor typical skin reactions to inoculation by theintradermal route, no adverse reactivity was seen in any of the monkeys.Small amounts of residual virus were isolated from skin lesions afterintradermal inoculation on days two and four post-inoculation only. Allspecimens were negative on day seven and later. There was no localreaction to intra-muscular injection. All four monkeys inoculated withALVAC-RG developed anti-rabies serum neutralizing antibodies as measuredin an RFFI test. Approximately six months after the initial inoculationall monkeys and one additional naive monkey were re-inoculated by thesubcutaneous route on the external face of the left thigh with 6.5 log₁₀TCID₅₀ of ALVAC-RG. Sera were analyzed for the presence of anti-rabiesantibody. The results are shown in Table 8.

Four of the five monkeys naive to rabies developed a serologicalresponse by seven days post-inoculation with ALVAC-RG. All five monkeyshad detectable antibody by 11 days post-inoculation. Of the four monkeyswith previous exposure to the rabies glycoprotein, all showed asignificant increase in serum neutralization titer between days 3 and 7post-vaccination. The results indicate that vaccination of squirrelmonkeys with ALVAC-RG does not produce adverse side-effects and aprimary neutralizing antibody response can be induced. An anamnesticresponse is also induced on re-vaccination. Prior exposure to ALVAC orto a canarypox recombinant expressing an unrelated foreign gene does notinterfere with induction of an anti-rabies immune response uponre-vaccination.

The immunological response of HIV-2 seropositive macaques to inoculationwith ALVAC-RG was assessed. Animals were inoculated as described aboveand the presence of anti-rabies serum neutralizing antibody assessed inan RFFI test. The results, shown in Table 9, indicated that HIV-2positive animals inoculated by the subcutaneous route developedanti-rabies antibody by 11 days after one inoculation. An anamnesticresponse was detected after a booster inoculation given approximatelythree months after the first inoculation. No response was detected inanimals receiving the recombinant by the oral route. In addition, aseries of six animals were inoculated with decreasing doses of ALVAC-RGgiven by either the intra-muscular or subcutaneous routes. Five of thesix animals inoculated responded by 14 days post-vaccination with nosignificant difference in antibody titer.

Two chimpanzees with prior exposure to HIV were inoculated with 7.0log₁₀ pfu of ALVAC-RG by the subcutaneous or intra-muscular route. At 3months post-inoculations both animals were re-vaccinated in an identicalfashion. The results are shown in Table 10.

No adverse reactivity to inoculation was noted by either intramuscularor subcutaneous routes. Both chimpanzees responded to primaryinoculation by 14 days and a strongly rising response was detectedfollowing re-vaccination.

TABLE 1 Sequential Passage of ALVAC in Avian and non-Avian Cells. CEFVero MRC5 Pass 1 Sample to^(a) 2.4 3.0 2.6 t7^(b) 7.0 1.4 0.4 t7A^(c)1.2 1.2 0.4 Pass 2 Sample to 5.0 0.4  N.D.^(d) t7 7.3 0.4 N.D. t7A 3.9N.D. N.D. Pass 3 Sample to 5.4 0.4 N.D. t7 7.4 N.D. N.D. t7A 3.8 N.D.N.D. Pass 4 Sample to 5.2 N.D. N.D. t7 7.1 N.D. N.D. t7A 3.9 N.D. N.D.^(a)This sample was harvested at zero time and represents the residualinput virus. The titer is expressed as log₁₀ pfu per ml. ^(b)This samplewas harvested at 7 days post-infection. ^(c)This sample was inoculatedin the presence of 40 μg/ml of Cytosine arabinoside and harvested at 7days post infection. ^(d)Not detectable

TABLE 2 Sequential Passage of ALVAC-RG in Avian and non-Avian Cells CEFVero MRC-5 Pass 1 Sample t0^(a) 3.0 2.9 2.9 t7^(b) 7.1 1.0 1.4 t7A^(c)1.8 1.4 1.2 Pass 2 Sample t0 5.1 0.4 0.4 t7 7.1  N.D.^(d) N.D. t7A 3.8N.D. N.D. Pass 3 Sample t0 5.1 0.4 N.D. t7 7.2 N.D. N.D. t7A 3.6 N.D.N.D. Pass 4 Sample t0 5.1 N.D. N.D. t7 7.0 N.D. N.D. t7A 4.0 N.D. N.D^(a)This sample was harvested at zero time and represents the residualinput virus. The titer is expressed as log₁₀ pfu per ml. ^(b)This samplewas harvested at 7 days post-infection. ^(c)This sample was inoculatedin the presence of 40 μg/ml of Cytosine arabinoside and harvested at 7days post-infection. ^(d)Not detectable.

TABLE 3 Amplification of residual virus by passage in CEF cells CEF VeroMRC-5 a) ALVAC Pass 2^(a)  7.0^(b) 6.0 5.2 3 7.5 4.1 4.9 4 7.5  N.D.^(c)N.D. 5 7.1 N.D. N.D. b) ALVAC-RG Pass 2^(a) 7.2 5.5 5.5 3 7.2 5.0 5.1 47.2 N.D. N.D. 5 7.2 N.D. N.D. ^(a)Pass 2 represents the amplification inCEF cells of the 7 day sample from Pass 1. ^(b)Titer expressed as log₁₀pfu per ml ^(c)Not Detectable

TABLE 4 Schedule of inoculation of rhesus macaques with ALVAC-RG (vCP65)Animal Inoculation 176L Primary: 1 × 10⁸ pfu of vCP65 orally in TANGSecondary: 1 × 10⁷ pfu of vCP65 plus 1 × 10⁷ pfu of vCP82^(a) by SCroute 185 L Primary: 1 × 10⁸ pfu of vCP65 orally in Tang Secondary: 1 ×10⁷ pfu of vCP65 plus 1 × 10⁷ pfu of vCP82 by SC route 177 L Primary: 5× 10⁷ pfu SC of vCP65 by SC route Secondary: 1 × 10⁷ pfu of vCP65 plus 1× 10⁷ pfu of vCP82 by SC route 186L Primary: 5 × 10⁷ pfu of vCP65 by SCroute Secondary: 1 × 10⁷ pfu of vCP65 plus 1 × 10⁷ pfu of vCP82 by SCroute 178L Primary: 1 × 10⁷ pfu of vCP65 by SC route 182L Primary: 1 ×10⁷ pfu of VCP65 by IM route 179L Primary: 1 × 10⁶ pfu of vCP65 by SCroute 183L Primary: 1 × 10⁶ pfu of vCP65 by IM route 180L Primary: 1 ×10⁶ pfu of vCP65 by SC route 184L Primary: 1 × 10⁵ pfu of vCP65 by IMroute 187L Primary 1 × 10⁷ pfu of vCP65 orally ^(a)vCP82 is a canarypoxvirus recombinant expressing the measles virus fusion and hemagglutiningenes.

TABLE 5 Analysis of yield in avian and non-avian cells inoculated withALVAC-RG Sample Time Cell Type t0 t72 t72A^(b) Expt 1 CEF  3.3^(a) 7.41.7 Vero 3.0 1.4 1.7 MRC-5 3.4 2.0 1.7 Expt 2 CEF 2.9 7.5 <1.7 WISH 3.32.2 2.0 Detroit-532 2.8 1.7 <1.7 ^(a)Titer expressed as log₁₀ pfu per ml^(b)Culture incubated in the presence of 40 μg/ml of Cytosinearabinoside

TABLE 6 Potency of ALVAC-RG as tested in mice Test Challenge Dose^(a)PD₅₀ ^(b) Initial seed 43 4.56 Primary seed 23 3.34 Vaccine Batch H 234.52 Vaccine Batch I 23 3.33 Vaccine Batch K 15 3.64 Vaccine Batch L 154.03 Vaccine Batch M 15 3.32 Vaccine Batch N 15 3.39 Vaccine Batch J 233.42 ^(a)Expressed as mouse LD₅₀ ^(b)Expressed as log₁₀ TCID₅₀

TABLE 7 Efficacy of ALVAC-RG in dogs and cats Dogs Cats DoseAntibody^(a) Survival^(b) Antibody Survival 6.7 11.9 5/5 2.9 3/5 7.710.1 N.T. 8.1 N.T. ^(a)Antibody at day 29 post inoculation expressed asthe geometric mean titer in International Units. ^(b)Expressed as aratio of survivors over animals challenged

TABLE 8 Anti-rabies serological response of Squirrel monkeys inoculatedwith canarypox recombinants Monkey Previous Rabies serum-neutralizingantibody^(a) # Exposure −196^(b) 0 3 7 11 21 28 22 ALVAC^(c)  NT^(g)<1.2 <1.2 <1.2 2.1 2.3 2.2 51 ALVAC^(c) NT <1.2 <1.2 1.7 2.2 2.2 2.2 39vCP37^(d) NT <1.2 <1.2 1.7 2.1 2.2 N.T.^(g) 55 vCP37^(d) NT <1.2 <1.21.7 2.2 2.1 N.T.  37 ALVAC-RG^(e) 2.2 <1.2 <1.2 3.2 3.5 3.5 3.2 53ALVAC-RG^(e) 2.2 <1.2 <1.2 3.6 3.6 3.6 3.4 38 ALVAC-RG^(f) 2.7 <1.7 <1.73.2 3.8 3.6 N.T.  54 ALVAC-RG^(f) 3.2 <1.7 <1.5 3.6 4.2 4.0 3.6 57 NoneNT <1.2 <1.2 1.7 2.7 2.7 2.3 ^(a)As determined by RFFI test on daysindicated and expressed in International Units ^(b)Day −196 representsserum from day 28 after primary vaccination ^(c)Animals received 5.0log₁₀ TCID₅₀ of ALVAC ^(d)Animals received 5.0 log₁₀ TCID₅₀ of vCP37^(e)Animals received 5.0 log₁₀ TCID₅₀ of ALVAC-RG ^(f)Animals received7.0 log₁₀ TCID₅₀ of ALVAC-RG ^(g)Not tested.

TABLE 9 Inoculation of rhesus macaques with ALVAC-RG^(a) Route ofPrimary Inoculation Days post- or/Tang SC SC SC IM SC IM SC IM ORInoculation 176L^(b) 185L 177L 186L 178L 182L 179L 183L 180L 184L187L^(b) −84  — — — −9 — — — — — —  3 — — — —  6 — — ± ± 11 — —  16^(d)128 19 — — 32 128 — — 35 — — 32 512 59 — — 64 256 75 — — 64 128 — — 99^(c) — — 64 256 — — — — — —  2 — — 32 256 — — — — — — —  6 — — 512 512 — — — — — — — 15 16 16 512  512 64 32 64 128 32 — — 29 16 32 256 256 64 64 32 128 32 — — 55 32 32 32 16 — 57 16 128  128 16 16 — ^(a)SeeTable 9 for schedule of inoculations. ^(b)Animals 176L and 185L received8.0 log₁₀ pfu by the oral route in 5 ml Tang. Animal 187L received 7.0log₁₀ pfu by oral route not in Tang. ^(c)Day of re-vaccination foranimals 176L, 185L, 177L and 186L by S.C. route, and primary vaccinationfor animals 178L, 182L, 179L, 183L, 180L, 184L and 187L. ^(d)Titersexpressed as reciprocal of last dilution showing inhibition offluorescence in an RFFI test.

TABLE 10 Inoculation of chimpanzees with ALVAC-RG Weeks post- Animal 431Animal 457 Inoculation I.M. S.C. 0  <8^(a) <8 1 <8 <8 2  8 32 4 16 32 816 32 12^(b)/0  16 8 13/1 128  128 15/3 256  512 20/8 64 128  26/12 32128 ^(a)Titer expressed as reciprocal of last dilution showinginhibition of fluorescence in an RFFI test ^(b)Day of re-inoculation

Example 10 Immunization of Humans Using Canarypox Expressing RabiesGlycoprotein (ALVAC-RG; vCP65)

ALVAC-RG (vCP65) was Generated as Described in Example 9 and FIGS. 9Aand 9B. For scaling-up and vaccine manufacturing ALVAC-RG (vCP65) wasgrown in primary CEF derived from specified pathogen free eggs. Cellswere infected at a multiplicity of 0.1 and incubated at 37° C. for threedays.

The vaccine virus suspension was obtained by ultrasonic disruption inserum free medium of the infected cells; cell debris were then removedby centrifugation and filtration. The resulting clarified suspension wassupplemented with lyophilization stabilizer (mixture of amino-acids),dispensed in single dose vials and freeze dried. Three batches ofdecreasing titer were prepared by ten-fold serial dilutions of the virussuspension in a mixture of serum free medium and lyophilizationstabilizer, prior to lyophilization.

Quality control tests were applied to the cell substrates, media andvirus seeds and final product with emphasis on the search foradventitious agents and inocuity in laboratory rodents. No undesirabletrait was found.

Preclinical data. Studies in vitro indicated that VERO or MRC-5 cells donot support the growth of ALVAC-RG (vCP65); a series of eight (VERO) and10 (MRC) blind serial passages caused no detectable adaptation of thevirus to grow in these non avian lines. Analyses of human cell lines(MRC-5, WISH, Detroit 532, HEL, HNK or EBV-transformed lymphoblastoidcells) infected or inoculated with ALVAC-RG (vCP65) showed noaccumulation of virus specific DNA suggesting that in these cells theblock in replication occurs prior to DNA synthesis. Significantly,however, the expression of the rabies virus glycoprotein gene in allcell lines tested indicating that the abortive step in the canarypoxreplication cycle occurs prior to viral DNA replication.

The safety and efficacy of ALVAC-RG (vCP65) were documented in a seriesof experiments in animals. A number of species including canaries,chickens, ducks, geese, laboratory rodents (suckling and adult mice),hamsters, guinea-pigs, rabbits, cats and dogs, squirrel monkeys, rhesusmacaques and chimpanzees, were inoculated with doses ranging from 10⁵ to10⁸ pfu. A variety of routes were used, most commonly subcutaneous,intramuscular and intradermal but also oral (monkeys and mice) andintracerebral (mice).

In canaries, ALVAC-RG (vCP65) caused a “take” lesion at the site ofscarification with no indication of disease or death. Intradermalinoculation of rabbits resulted in a typical poxvirus inoculationreaction which did not spread and healed in seven to ten days. There wasno adverse side effects due to canarypox in any of the animal tests.Immunogenicity was documented by the development of anti-rabiesantibodies following inoculation of ALVAC-RG (vCP65) in rodents, dogs,cats, and primates, as measured by Rapid Fluorescent Focus InhibitionTest (RFFIT). Protection was also demonstrated by rabies virus challengeexperiments in mice, dogs, and cats immunized with ALVAC-RG (vCP65).

Volunteers. Twenty-five healthy adults aged 20-45 with no previoushistory of rabies immunization were enrolled. Their health status wasassessed by complete medical histories, physical examinations,hematological and blood chemistry analyses. Exclusion criteria includedpregnancy, allergies, immune depression of any kind, chronicdebilitating disease, cancer, injection of immunoglobins in the pastthree months, and seropositivity to human immunodeficiency virus (HIV)or to hepatitis B virus surface antigen.

Study design. Participants were randomly allocated to receive eitherstandard Human Diploid Cell Rabies Vaccine (HDC) batch no E0751 (PasteurMerieux Serums & Vaccine, Lyon, France) or the study vaccine ALVAC-RG(vCP65).

The trial was designated as a dose escalation study. Three batches ofexperimental ALVAC-RG (vCP65) vaccine were used sequentially in threegroups of volunteers (Groups A, B and C) with two week intervals betweeneach step. The concentration of the three batches was 10^(3.5),10^(4.5), 10^(5.5) Tissue Culture Infectious Dose (TCID₅₀) per dose,respectively.

Each volunteer received two doses of the same vaccine subcutaneously inthe deltoid region at an interval of four weeks. The nature of theinjected vaccine was not known by the participants at the time of thefirst injection but was known by the investigator.

In order to minimize the risk of immediate hypersensitivity at the timeof the second injection, the volunteers of Group B allocated to themedium dose of experimental vaccine were injected 1 h previously withthe lower dose and those allocated to the higher dose (Group C) receivedsuccessively the lower and the medium dose at hourly intervals.

Six months later, the recipients of the highest dosage of ALVAC-RG(vCP65) (Group C) and HDC vaccine were offered a third dose of vaccine;they were then randomized to receive either the same vaccine aspreviously or the alternate vaccine. As a result, four groups wereformed corresponding to the following immunization scheme: 1. HDC,HDC-HDC; 2. HDC, HDC-ALVAC-RG (vCP65); 3. ALVAC-RG (vCP65), ALVAC-RG(vCP65)-HDC; 4. ALVAC-RG (vCP65), ALVAC-RG (vCP65), ALVAC-RG (vCP65).

Monitoring of Side Effects. All subjects were monitored for 1 h afterinjection and re-examined every day for the next five days. They wereasked to record local and systemic reactions for the next three weeksand were questioned by telephone two times a week.

Laboratory Investigators. Blood specimens were obtained beforeenrollment and two, four and six days after each injection. Analysisincluded complete blood cell count, liver enzymes and creatine kinaseassays.

Antibody assays. Antibody assays were performed seven days prior to thefirst injection and at days 7, 28, 35, 56, 173, 187 and 208 of thestudy.

The levels of neutralizing antibodies to rabies were determined usingthe Rapid Fluorescent Focus Inhibition test (RFFIT) (Smith et al.,1973). Canarypox antibodies were measured by direct ELISA. The antigen,a suspension of purified canarypox virus disrupted with 0.1% TritonX100, was coated in microplates. Fixed dilutions of the sera werereacted for two hours at room temperature and reacting antibodies wererevealed with a peroxidase labelled anti-human IgG goat serum. Theresults are expressed as the optical density read at 490 nm.

Analysis. Twenty-five subjects were enrolled and completed the study.There were 10 males and 15 females and the mean age was 31.9 (21 to 48).All but three subjects had evidence of previous smallpox vaccination;the three remaining subjects had no typical scar and vaccinationhistory. Three subjects received each of the lower doses of experimentalvaccine (10^(3.5) and 10^(4.5) TCID₅₀), nine subjects received 10^(5.5)TCID₅₀ and ten received the HDC vaccine.

Safety (Table 11). During the primary series of immunization, fevergreater than 37.7° C. was noted within 24 hours after injection in oneHDC recipient (37.8° C.) and in one vCP65 10^(5.5) TCID₅₀ recipient (38°C.). No other systemic reaction attributable to vaccination was observedin any participant.

Local reactions were noted in 9/10 recipients of HDC vaccine injectedsubcutaneously and in 0/3, 1/3 and 9/9 recipients of vCP65 10^(3.5),10^(4.5), 10^(5.5) TCID₅₀, respectively.

Tenderness was the most common symptoms and was always mild. Other localsymptoms included redness and induration which were also mild andtransient. All symptoms usually subsided within 24 hours and neverlasted more than 72 hours.

There was no significant change in blood cell counts, liver enzymes orcreatine kinase values.

Immune Responses; Neutralizing Antibodies to Rabies (Table 12). Twentyeight days after the first injection all the HDC recipients hadprotective titers (≧0.5 IU/ml). By contrast none in groups A and B(10^(3.5) and 10^(4.5) TCID₅₀) and only 2/9 in group C (10^(5.5) TCID₅₀)ALVAC-RG (vCP65) recipients reached this protective titer.

At day 56 (i.e. 28 days after the second injection) protective titerswere achieved in 0/3 of Group A, 2/3 of Group B and 9/9 of Group Crecipients of ALVAC-RG (vCP65) vaccine and persisted in all 10 HDCrecipients.

At day 56 the geometric mean titers were 0.05, 0.47, 4.4 and 11.5 IU/mlin groups A, B, C and HDC respectively.

At day 180, the rabies antibody titers had substantially decreased inall subjects but remained above the minimum protective titer of 0.5IU/ml in 5/10 HCD recipients and in 5/9 ALVAC-RG (vCP65) recipients; thegeometric mean titers were 0.51 and 0.45 IU/ml in groups HCD and C,respectively.

Antibodies to the Canarypox virus (Table 13). The pre-immune titersobserved varied widely with titers varying from 0.22 to 1.23 O.D. unitsdespite the absence of any previous contact with canary birds in thosesubjects with the highest titers. When defined as a greater thantwo-fold increase between preimmunization and post second injectiontiters, a seroconversion was obtained in 1/3 subjects in group B and in9/9 subjects in group C whereas no subject seroconverted in groups A orHDC.

Booster Injection. The vaccine was similarly well tolerated six monthslater, at the time of the booster injection: fever was noted in 2/9 HDCbooster recipients and in 1/10 ALVAC-RG (vCP65) booster recipients.Local reactions were present in 5/9 recipients of HDC booster and in6/10 recipients of the ALVAC-RG (vCP65) booster.

Observations. FIGS. 13A-13D show graphs of rabies neutralizing antibodytiters (Rapid Fluorescent Focus Inhibition Test or RFFIT, IU/ml):Booster effect of HDC and vCP65 (10^(5.5) TCID₅₀) in volunteerspreviously immunized with either the same or the alternate vaccine.Vaccines were given at days 0, 28 and 180. Antibody titers were measuredat days 0, 7, 28, 35, 56, 173, and 187 and 208.

As shown in FIGS. 13A to 13D, the booster dose given resulted in afurther increase in rabies antibody titers in every subject whatever theimmunization scheme. However, the ALVAC-RG (vCP65) booster globallyelicited lower immune responses than the HDC booster and the ALVAC-RG(vCP65), ALVAC-RG (vCP65)-ALVAC-RG (vCP65) group had significantly lowertiters than the three other groups. Similarly, the ALVAC-RG (vCP65)booster injection resulted in an increase in canarypox antibody titersin 3/5 subjects who had previously received the HDC vaccine and in allfive subjects previously immunized with ALVAC-RG (vCP65).

In general, none of the local side effects from administration of vCP65was indicative of a local replication of the virus. In particular,lesions of the skin such as those observed after injection of vaccinewere absent. In spite of the apparent absence of replication of thevirus, the injection resulted in the volunteers generating significantamounts of antibodies to both the canarypox vector and to the expressedrabies glycoprotein.

Rabies neutralizing antibodies were assayed with the Rapid FluorescentFocus Inhibition Test (RFFIT) which is known to correlate well with thesero neutralization test in mice. Of 9 recipients of 10^(5.5)TCID₅₀,five had low level responses after the first dose. Protective titers ofrabies antibodies were obtained after the second injection in allrecipients of the highest dose tested and even in 2 of the 3 recipientsof the medium dose. In this study, both vaccines were givensubcutaneously as usually recommended for live vaccines, but not for theinactivated HDC vaccine. This route of injection was selected as it bestallowed a careful examination of the injection site, but this couldexplain the late appearance of antibodies in HDC recipients: indeed,none of the HDC recipients had an antibody increase at day 7, whereas,in most studies where HDC vaccine is give intramuscularly a significantproportion of subjects do (Klietmann et al., Int'l Green Cross—Geneva,1981; Kuwert et al., Int'l Green Cross—Geneva, 1981). However, thisinvention is not necessarily limited to the subcutaneous route ofadministration.

The GMT (geometric mean titers) of rabies neutralizing antibodies waslower with the investigational vaccine than with the HDC controlvaccine, but still well above the minimum titer required for protection.The clear dose effect response obtained with the three dosages used inthis study suggest that a higher dosage might induce a strongerresponse. Certainly from this disclosure the skilled artisan can selectan appropriate dosage for a given patient.

The ability to boost the antibody response is another important resultof this Example; indeed, an increase in rabies antibody titers wasobtained in every subject after the 6 month dose whatever theimmunization scheme, showing that preexisting immunity elicited byeither the canarypox vector or the rabies glycoprotein had no blockingeffect on the booster with the recombinant vaccine candidate or theconventional HDC rabies vaccine. This contrasts findings of others withvaccinia recombinants in humans that immune response may be blocked bypre-existing immunity (Cooney et al.; Etinger et al.).

Thus, this Example clearly demonstrates that a non-replicating poxviruscan serve as an immunizing vector in humans, with all of the advantagesthat replicating agents confer on the immune response, but without thesafety problem created by a fully permissive virus.

TABLE 11 Reactions in the 5 days following vaccination vCP65 dosage H DC (TCID50) 10^(3.5) 10^(4.5) 10^(5.5) control Injection 1st 2nd 1st 2nd1st 2nd 1st 2nd No. vaccinees 3 3 3 3 9 9 10 10 temp >37.7° C. 0 0 0 0 01 1 0 soreness 0 0 1 1 6 8 8 6 redness 0 0 0 0 0 4 5 4 induration 0 0 00 0 4 5 4

TABLE 12 Rabies neutralizing antibodies (REFIT; IU/ml) Individual titersand geometric mean titers (GMT) Days No. TCID50/dose 0 7 28 35 56 110^(3.5) <0.1 <0.1 <0.1 <0.1 0.2 3 10^(3.5) <0.1 <0.1 <0.1 <0.1 <0.1 410^(3.5) <0.1 <0.1 <0.1 <0.1 <0.1 G.M.T. <0.1 <0.1 <0.1 <0.1 <0.1 610^(4.5) <0.1 <0.1 <0.1 <0.1 <0.1 7 10^(4.5) <0.1 <0.1 <0.1 2.4 1.9 1010^(4.5) <0.1 <0.1 <0.1 1.6 1.1 G.M.T. <0.1 <0.1 0.1 0.58 0.47 1110^(5.5) <0.1 <0.1 1.0 3.2 4.3 13 10^(5.5) <0.1 <0.1 0.3 6.0 8.8 1410^(5.5) <0.1 <0.1 0.2 2.1 9.4 17 10^(5.5) <0.1 <0.1 <0.1 1.2 2.5 1810^(5.5) <0.1 <0.1 0.7 8.3 12.5 20 10^(5.5) <0.1 <0.1 <0.1 0.3 3.7 2110^(5.5) <0.1 <0.1 0.2 2.6 3.9 23 10^(5.5) <0.1 <0.1 <0.1 1.7 4.2 2510^(5.5) <0.1 <0.1 <0.1 0.6 0.9 G.M.T. <0.1 <0.1 0.16 1.9 4.4* 2 HDC<0.1 <0.1 0.8 7.1 7.2 5 HDC <0.1 <0.1 9.9 12.8 18.7 8 HDC <0.1 <0.1 12.721.1 16.5 9 HDC <0.1 <0.1 6.0 9.9 14.3 12 HDC <0.1 <0.1 5.0 9.2 25.3 15HDC <0.1 <0.1 2.2 5.2 8.6 16 HDC <0.1 <0.1 2.7 7.7 20.7 19 HDC <0.1 <0.12.6 9.9 9.1 22 HDC <0.1 <0.1 1.4 8.6 6.6 24 HDC <0.1 <0.1 0.8 5.8 4.7G.M.T. <0.1 <0.1 2.96 9.0 11.5* *p = 0.007 student t test

TABLE 13 Canarypox antibodies: ELISA Geometric Mean Titers* vCP65 dosageDays TCID50/dose 0 7 28 35 56 10^(3.5) 0.69 ND 0.76 ND 0.68 10^(4.5)0.49 0.45 0.56 0.63 0.87 10^(5.5) 0.38 0.38 0.77 1.42 1.63 HDC control0.45 0.39 0.40 0.35 0.39 *optical density at 1/25 dilution

Example 11 Comparison of the LD₅₀ of ALVAC and NYVAC with VariousVaccinia Virus Strains

Mice. Male outbred Swiss Webster mice were purchased from Taconic Farms(Germantown, N.Y.) and maintained on mouse chow and water ad libitumuntil use at 3 weeks of age (“normal” mice). Newborn outbred SwissWebster mice were of both sexes and were obtained following timedpregnancies performed by Taconic Farms. All newborn mice used weredelivered within a two day period.

Viruses. ALVAC was derived by plaque purification of a canarypox viruspopulation and was prepared in primary chick embryo fibroblast cells(CEF). Following purification by centrifugation over sucrose densitygradients, ALVAC was enumerated for plaque forming units in CEF cells.The WR(L) variant of vaccinia virus was derived by selection of largeplaque phenotypes of WR (Panicali et al., 1981). The Wyeth New YorkState Board of Health vaccine strain of vaccinia virus was obtained fromPharmaceuticals Calf Lymph Type vaccine Dryvax, control number 302001B.Copenhagen strain vaccinia virus VC-2 was obtained from InstitutMerieux, France. Vaccinia virus strain NYVAC was derived from CopenhagenVC-2. All vaccinia virus strains except the Wyeth strain were cultivatedin Vero African green monkey kidney cells, purified by sucrose gradientdensity centrifugation and enumerated for plaque forming units on Verocells. The Wyeth strain was grown in CEF cells and enumerated for plaqueforming units in CEF cells.

Inoculations. Groups of 10 normal mice were inoculated intracranially(ic) with 0.05 ml of one of several dilutions of virus prepared by10-fold serially diluting the stock preparations in sterilephosphate-buffered saline. In some instances, undiluted stock viruspreparation was used for inoculation.

Groups of 10 newborn mice, 1 to 2 days old, were inoculated ic similarlyto the normal mice except that an injection volume of 0.03 ml was used.

All mice were observed daily for mortality for a period of 14 days(newborn mice) or 21 days (normal mice) after inoculation. Mice founddead the morning following inoculation were excluded due to potentialdeath by trauma.

The lethal dose required to produce mortality for 50% of theexperimental population (LD₅₀) was determined by the proportional methodof Reed and Muench.

Comparison of the LD₅₀ of ALVAC and NYVAC with Various Vaccinia VirusStrains for Normal, Young Outbred Mice by the ic Route. In young, normalmice, the virulence of NYVAC and ALVAC were several orders of magnitudelower than the other vaccinia virus strains tested (Table 14). NYVAC andALVAC were found to be over 3,000 times less virulent in normal micethan the Wyeth strain; over 12,500 times less virulent than the parentalVC-2 strain; and over 63,000,000 times less virulent than the WR(L)variant. These results would suggest that NYVAC is highly attenuatedcompared to other vaccinia strains, and that ALVAC is generallynonvirulent for young mice when administered intracranially, althoughboth may cause mortality in mice at extremely high doses (3.85×10⁸ PFUs,ALVAC and 3×10⁸ PFUs, NYVAC) by an undetermined mechanism by this routeof inoculation.

Comparison of the LD₅₀ of ALVAC and NYVAC with Various Vaccinia VirusStrains for Newborn Outbred Mice by the ic Route. The relative virulenceof 5 poxvirus strains for normal, newborn mice was tested by titrationin an intracranial (ic) challenge model system (Table 15). Withmortality as the endpoint, LD₅₀ values indicated that ALVAC is over100,000 times less virulent than the Wyeth vaccine strain of vacciniavirus; over 200,000 times less virulent than the Copenhagen VC-2 strainof vaccinia virus; and over 25,000,000 times less virulent than the WR-Lvariant of vaccinia virus. Nonetheless, at the highest dose tested,6.3×10⁷ PFUs, 100% mortality resulted. Mortality rates of 33.3% wereobserved at 6.3×10⁶ PFUs. The cause of death, while not actuallydetermined, was not likely of toxicological or traumatic nature sincethe mean survival time (MST) of mice of the highest dosage group(approximately 6.3 LD₅₀) was 6.7±1.5 days. When compared to WR(L) at achallenge dose of 5 LD₅₀, wherein MST is 4.8±0.6 days, the MST of ALVACchallenged mice was significantly longer (P=0.001).

Relative to NYVAC, Wyeth was found to be over 15,000 times morevirulent; VC-2, greater than 35,000 times more virulent; and WR(L), over3,000,000 times more virulent. Similar to ALVAC, the two highest dosesof NYVAC, 6×10⁸ and 6×10⁷ PFUs, caused 100% mortality. However, the MSTof mice challenged with the highest dose, corresponding to 380 LD₅₀, wasonly 2 days (9 deaths on day 2 and 1 on day 4). In contrast, all micechallenged with the highest dose of WR-L, equivalent to 500 LD₅₀,survived to day 4.

TABLE 14 Calculated 50% Lethal Dose for mice by various vaccinia virusstrains and for canarypox virus (ALVAC) by the ic route. POXVIRUSCALCULATED STRAIN LD₅₀ (PFUs) WR(L) 2.5 VC-2 1.26 × 10⁴ WYETH 5.00 × 10⁴NYVAC 1.58 × 10⁸ ALVAC 1.58 × 10⁸

TABLE 15 Calculated 50% Lethal Dose for newborn mice by various vacciniavirus strains and for canarypox virus (ALVAC) by the ic route. POXVIRUSCALCULATED STRAIN LD₅₀ (PFUs) WR(L) 0.4 VC-2 0.1 WYETH 1.6 NYVAC 1.58 ×10⁶ ALVAC 1.00 × 10⁷

Example 12 Evaluation of NYVAC (vP866) and NYVAC-RG (vP879)

Immunoprecipitations. Preformed monolayers of avian or non-avian cellswere inoculated with 10 pfu per cell of parental NYVAC (vP866) orNYVAC-RG (vP879) virus. The inoculation was performed in EMEM free ofmethionine and supplemented with 2% dialyzed fetal bovine serum. After aone hour incubation, the inoculum was removed and the medium replacedwith EMEM (methionine free) containing 20 μCi/ml of ³⁵S-methionine.After an overnight incubation of approximately 16 hours, cells werelysed by the addition of Buffer A (1% Nonidet P-40, 10 mM Tris pH7.4,150 mM NaCl, 1 mM EDTA, 0.01% sodium azide, 500 units per ml ofaprotinin, and 0.02% phenyl methyl sulfonyl fluoride).Immunoprecipitation was performed using a rabies glycoprotein specificmonoclonal antibody designated 24-3F10 supplied by Dr. C. Trinarchi,Griffith Laboratories, New York State Department of Health, Albany,N.Y., and a rat anti-mouse conjugate obtained from Boehringer MannheimCorporation (Cat. #605-500). Protein A Sepharose CL-48 obtained fromPharmacia LKB Biotechnology Inc., Piscataway, N.J., was used as asupport matrix. Immunoprecipitates were fractionated on 10%polyacrylamide gels according to the method of Dreyfuss et. al. (1984).Gels were fixed, treated for fluorography with 1M Na-salicylate for onehour, and exposed to Kodak XAR-2 film to visualize theimmunoprecipitated protein species.

Sources of Animals. New Zealand White rabbits were obtained fromHare-Marland (Hewitt, N.J.). Three week old male Swiss Webster outbredmice, timed pregnant female Swiss Webster outbred mice, and four weekold Swiss Webster nude (nu⁺nu⁺) mice were obtained from Taconic Farms,Inc. (Germantown, N.Y.). All animals were maintained according to NIHguidelines. All animal protocols were approved by the institutionalIACUC. When deemed necessary, mice which were obviously terminally illwere euthanized.

Evaluation of Lesions in Rabbits. Each of two rabbits was inoculatedintradermally at multiple sites with 0.1 ml of PBS containing 10⁴, 10⁵,10⁶, 10⁷, or 10⁸ pfu of each test virus or with PBS alone. The rabbitswere observed daily from day 4 until lesion resolution. Indurations andulcerations were measured and recorded.

Virus Recovery from Inoculation Sites. A single rabbit was inoculatedintradermally at multiple sites of 0/1 ml of PBS containing 10⁶, 10⁷, or10⁸ pfu of each test virus or with PBS alone. After 11 days, the rabbitwas euthanized and skin biopsy specimens taken from each of theinoculation sites were aseptically prepared by mechanical disruption andindirect sonication for virus recovery. Infectious virus was assayed byplaque titration on CEF monolayers.

Virulence in Mice. Groups of ten mice, or five in the nude miceexperiment, were inoculated ip with one of several dilutions of virus in0.5 ml of sterile PBS. Reference is also made to Example 11.

Cyclophosphamide (CY) Treatment. Mice were injected by the ip route with4 mg (0.02 ml) of CY (SIGMA) on day −2, followed by virus injection onday 0. On the following days post infection, mice were injected ip withCY: 4 mg on day 1; 2 mg on days 4, 7 and 11; 3 mg on days 14, 18, 21, 25and 28. Immunosuppression was indirectly monitored by enumerating whiteblood cells with a Coulter Counter on day 11. The average white bloodcell count was 13,500 cells per μl for untreated mice (n=4) and 4,220cells per μl for CY-treated control mice (n=5).

Calculation of LD₅₀. The lethal dose required to produce 50% mortality(LD₅₀) was determined by the proportional method of Reed and Muench(Reed and Muench 1938).

Potency Testing of NYVAC-RG in Mice. Four to six week old mice wereinoculated in the footpad with 50 to 100 μl of a range of dilutions(2.0-8.0 log₁₀ tissue culture infective dose 50% (TCID₅₀)) of eitherVV-RG (Kieny et al., 1984), ALVAC-RG (Taylor et al., 1991b), or theNYVAC-RG. Each group consisted of eight mice. At 14 dayspost-vaccination, the mice were challenged by intracranial inoculationwith 15 LD₅₀ of the rabies virus CVS strain (0.03 ml). On day 28,surviving mice were counted and protective does 50% (PD₅₀) calculated.

Derivation of NYVAC (vP866). The NYVAC strain of vaccinia virus wasgenerated from VC-2, a plaque cloned isolate of the COPENHAGEN vaccinestrain. To generate NYVAC from VC-2, eighteen vaccinia ORFs, including anumber of viral functions associated with virulence, were preciselydeleted in a series of sequential manipulations as described earlier inthis disclosure. These deletions were constructed in a manner designedto prevent the appearance of novel unwanted open reading frames. FIG. 10schematically depicts the ORFs deleted to generate NYVAC. At the top ofFIG. 10 is depicted the HindIII restriction map of the vaccinia virusgenome (VC-2 plaque isolate, COPENHAGEN strain). Expanded are the sixregions of VC-2 that were sequentially deleted in the generation ofNYVAC. The deletions were described earlier in this disclosure (Examples1 through 6). Below such deletion locus is listed the ORFs which weredeleted from that locus, along with the functions or homologies andmolecular weight of their gene products.

Replication Studies of NYVAC and ALVAC on Human Tissue Cell Lines. Inorder to determine the level of replication of NYVAC strain of vacciniavirus (vP866) in cells of human origin, six cell lines were inoculatedat an input multiplicity of 0.1 pfu per cell under liquid culture andincubated for 72 hours. The COPENHAGEN parental clone (VC-2) wasinoculated in parallel. Primary chick embryo fibroblast (CEF) cells(obtained from 10-11 day old embryonated eggs of SPF origin, Spafas,Inc., Storrs, Conn.) were included to represent a permissive cellsubstrate for all viruses. Cultures were analyzed on the basis of twocriteria: the occurrence of productive viral replication and expressionof an extrinsic antigen.

The replication potential of NYVAC in a number of human derived cellsare shown in Table 16. Both VC-2 and NYVAC are capable of productivereplication in CEF cells, although NYVAC with slightly reduced yields.VC-2 is also capable of productive replication in the six human derivedcell lines tested with comparable yields except in the EBV transformedlymphoblastoid cell line JT-1 (human lymphoblastoid cell linetransformed with Epstein-Barr virus, see Rickinson et al., 1984). Incontrast, NYVAC is highly attenuated in its ability to productivelyreplicate in any of the human derived cell lines tested. Small increasesof infectious virus above residual virus levels were obtained fromNYVAC-infected MRC-5 (ATCC #CCL171, human embryonic lung origin),DETROIT 532 (ATCC #CCL54, human foreskin, Downs Syndrome), HEL 299 (ATCC#CCL137, human embryonic lung cells) and HNK (human neonatal kidneycells, Whittiker Bioproducts, Inc. Walkersville, Md., Cat #70-151)cells. Replication on these cell lines was significantly reduced whencompared to virus yields obtained from NYVAC-infected CEF cells or withparental VC-2 (Table 16). It should be noted that the yields at 24 hoursin CEF cells for both NYVAC and VC-2 is equivalent to the 72-hour yield.Allowing the human cell line cultures to incubate an additional 48 hours(another two viral growth cycles) may, therefore, have amplified therelative virus yield obtained.

Consistent with the low levels of virus yields obtained in thehuman-derived cell lines, MRC-5 and DETROIT 532, detectable but reducedlevels of NYVAC-specific DNA accumulation were noted. The level of DNAaccumulation in the MRC-5 and DETROIT 532 NYVAC-infected cell linesrelative to that observed in NYVAC-infected CEF cells paralleled therelative virus yields. NYVAC-specific viral DNA accumulation was notobserved in any of the other human-derived cells.

An equivalent experiment was also performed using the avipox virus,ALVAC. The results of virus replication are also shown in Table 16. Noprogeny virus was detectable in any of the human cell lines consistentwith the host range restriction of canarypox virus to avian species.Also consistent with a lack of productive replication of ALVAC in thesehuman-derived cells is the observation that no ALVAC-specific DNAaccumulation was detectable in any of the human-derived cell lines.

Expression of Rabies Glycoprotein by NYVAC-RG (vP879) in Human Cells. Inorder to determine whether efficient expression of a foreign gene couldbe obtained in the absence of significant levels of productive viralreplication, the same cell lines were inoculated with the NYVACrecombinant expressing the rabies virus glycoprotein (vP879, Example 7)in the presence of ³⁵S-methionine. Immunoprecipitation of the rabiesglycoprotein was performed from the radiolabelled culture lysate using amonoclonal antibody specific for the rabies glycoprotein.Immunoprecipitation of a 67 kDa protein was detected consistent with afully glycosylated form of the rabies glycoprotein. No serologicallycrossreactive product was detected in uninfected or parental NYVACinfected cell lysates. Equivalent results were obtained with all otherhuman cells analyzed.

Inoculations on the Rabbit Skin. The induction and nature of skinlesions on rabbits following intradermal (id) inoculations has beenpreviously used as a measure of pathogenicity of vaccinia virus strains(Buller et al., 1988; Child et al., 1990; Fenner, 1958, Flexner et al.,1987; Ghendon and Chemos 1964). Therefore, the nature of lesionsassociated with id inoculations with the vaccinia strains WR (ATCC#VR119 plaque purified on CV-1 cells, ATCC #CCL70, and a plaque isolatedesignated L variant, ATCC #VR2035 selected, as described in Panicali etal., 1981)), WYETH (ATCC #VR325 marketed as DRYVAC by WyethLaboratories, Marietta, Pa.), COPENHAGEN (VC-2), and NYVAC was evaluatedby inoculation of two rabbits (A069 and A128). The two rabbits displayeddifferent overall sensitivities to the viruses, with rabbit A128displaying less severe reactions than rabbit A069. In rabbit A128,lesions were relatively small and resolved by 27 days post-inoculation.On rabbit A069, lesions were intense, especially for the WR inoculationsites, and resolved only after 49 days. Intensity of the lesions wasalso dependent on the location of the inoculation sites relative to thelymph drainage network. In particular, all sites located above thebackspine displayed more intense lesions and required longer times toresolve the lesions located on the flanks. All lesions were measureddaily from day 4 to the disappearance of the last lesion, and the meansof maximum lesion size and days to resolution were calculated (Table17). No local reactions were observed from sites injected with thecontrol PBS. Ulcerative lesions were observed at sites injected with WR,VC-2 and WYETH vaccinia virus strains. Significantly, no induration orulcerative lesions were observed at sites of inoculation with NYVAC.

Persistence of Infectious Virus at the Site of Inoculation. To assessthe relative persistence of these viruses at the site of inoculation, arabbit was inoculated intradermally at multiple sites with 0.1 ml PBScontaining 10⁶, 10⁷ or 10⁸ pfu of VC-2, WR, WYETH or NYVAC. For eachvirus, the 10⁷ pfu dose was located above the backspine, flanked by the10⁶ and 10⁸ doses. Sites of inoculation were observed daily for 11 days.WR elicited the most intense response, followed by VC-2 and WYETH (Table18). Ulceration was first observed at day 9 for WR and WYETH and day 10for VC-2. Sites inoculated with NYVAC or control PBS displayed noinduration or ulceration. At day 11 after inoculation, skin samples fromthe sites of inoculation were excised, mechanically disrupted, and viruswas titrated on CEF cells. The results are shown in Table 18. In no casewas more virus recovered at this timepoint than was administered.Recovery of vaccinia strain, WR, was approximately 10⁶ pfu of virus ateach site irrespective of amount of virus administered. Recovery ofvaccinia strains WYETH and VC-2 was 10³ to 10⁴ pfu regardless of amountadministered. No infectious virus was recovered from sites inoculatedwith NYVAC.

Inoculation of Genetically or Chemically Immune Deficient Mice.Intraperitoneal inoculation of high doses of NYVAC (5×10⁸ pfu) or ALVAC(10⁹ pfu) into nude mice caused no deaths, no lesions, and no apparentdisease through the 100 day observation period. In contrast, miceinoculated with WR (10³ to 10⁴ pfu), WYETH (5×10⁷ or 5×10⁸ pfu) or VC-2(10⁴ to 10⁹ pfu) displayed disseminated lesions typical of poxvirusesfirst on the toes, then on the tail, followed by severe orchitis in someanimals. In mice infected with WR or WYETH, the appearance ofdisseminated lesions generally led to eventual death, whereas most miceinfected with VC-2 eventually recovered. Calculated LD₅₀ values aregiven in Table 19.

In particular, mice inoculated with VC-2 began to display lesions ontheir toes (red papules) and 1 to 2 days later on the tail. Theselesions occurred between 11 and 13 days post-inoculation (pi) in micegiven the highest doses (10⁹, 10⁸, 10⁷ and 10⁶ pfu), on day 16 pi inmice given 10⁵ pfu and on day 21 pi in mice given 10⁴ pfu. No lesionswere observed in mice inoculated with 10³ and 10² pfu during the 100 dayobservation period. Orchitis was noticed on day 23 pi in mice given 10⁹and 10⁸ pfu, and approximately 7 days later in the other groups (10⁷ to10⁴ pfu). Orchitis was especially intense in the 10⁹ and 10⁸ pfu groupsand, although receding, was observed until the end of the 100 dayobservation period. Some pox-like lesions were noticed on the skin of afew mice, occurring around 30-35 days pi. Most pox lesions healednormally between 60-90 days pi. Only one mouse died in the groupinoculated with 10⁹ pfu (Day 34 pi) and one mouse died in the groupinoculated with 10⁸ pfu (Day 94 pi). No other deaths were observed inthe VC-2 inoculated mice.

Mice inoculated with 10⁴ pfu of the WR strain of vaccinia started todisplay pox lesions on Day 17 pi. These lesions appeared identical tothe lesions displayed by the VC-2 injected mice (swollen toes, tail).Mice inoculated with 10³ pfu of the WR strain did not develop lesionsuntil 34 days pi. Orchitis was noticed only in the mice inoculated withthe highest dose of WR (10⁴pfu). During the latter stages of theobservation period, lesions appeared around the mouth and the micestopped eating. All mice inoculated with 10⁴ pfu of WR died or wereeuthanized when deemed necessary between 21 days and 31 days pi. Fourout of the 5 mice injected with 10³ pfu of WR died or were euthanizedwhen deemed necessary between 35 days and 57 days pi. No deaths wereobserved in mice inoculated with lower doses of WR (1 to 100 pfu).

Mice inoculated with the WYETH strain of vaccinia virus at higher doses5×10⁷ and 5×10⁸ pfu) showed lesions on toes and tails, developedorchitis, and died. Mice injected with 5×10⁶ pfu or less of WYETH showedno signs of disease or lesions.

As shown in Table 19, CY-treated mice provided a more sensitive modelfor assaying poxvirus virulence than did nude mice. LD₅₀ values for theWR, WYETH, and VC-2 vaccinia virus strains were significantly lower inthis model system than in the nude mouse model. Additionally, lesionsdeveloped in mice injected with WYETH, WR and VC-2 vaccinia viruses, asnoted below, with higher doses of each virus resulting in more rapidformation of lesions. As was seen with nude mice, CY-treated miceinjected with NYVAC or ALVAC did not develop lesions. However, unlikenude mice, some deaths were observed in CY-treated mice challenged withNYVAC or ALVAC, regardless of the dose. These random incidences aresuspect as to the cause of death.

Mice injected with all doses of WYETH (9.5×10⁴ to 9.5×10⁹ pfu) displayedpox lesions on their tail and/or on their toes between 7 and 15 days pi.In addition, the tails and toes were swollen. Evolution of lesions onthe tail was typical of pox lesions with formation of a papule,ulceration and finally formation of a scab. Mice inoculated with alldoses of VC-2 (1.65×10⁵ to 1.65×10⁹) also developed pox lesions on theirtails and/or their toes analogous to those of WYETH injected mice. Theselesions were observed between 7-12 days post inoculation. No lesionswere observed on mice injected with lower doses of WR virus, althoughdeaths occurred in these groups.

Potency Testing of NYVAC-RG. In order to determine that attenuation ofthe COPENHAGEN strain of vaccinia virus had been effected withoutsignificantly altering the ability of the resulting NYVAC strain to be auseful vector, comparative potency tests were performed. In order tomonitor the immunogenic potential of the vector during the sequentialgenetic manipulations performed to attenuate the virus, a rabiesvirusglycoprotein was used as a reporter extrinsic antigen. The protectiveefficacy of the vectors expressing the rabies glycoprotein gene wasevaluated in the standard NIH mouse potency test for rabies (Seligmann,1973). Table 20 demonstrates that the PD₅₀ values obtained with thehighly attenuated NYVAC vector are identical to those obtained using aCOPENHAGEN-based recombinant containing the rabies glycoprotein gene inthe tk locus (Kieny et al., 1984) and similar to PD₅₀ values obtainedwith ALVAC-RG, a canarypox based vector restricted to replication toavian species.

Observations. NYVAC, deleted of known virulence genes and havingrestricted in vitro growth characteristics, was analyzed in animal modelsystems to assess its attenuation characteristics. These studies wereperformed in comparison with the neurovirulent vaccinia virus laboratorystrain, WR, two vaccinia virus vaccine strains, WYETH (New York CityBoard of Health) and COPENHAGEN (VC-2), as well as with a canarypoxvirus strain, ALVAC (See also Example 11). Together, these virusesprovided a spectrum of relative pathogenic potentials in the mousechallenge model and the rabbit skin model, with WR being the mostvirulent strain, WYETH and COPENHAGEN (VC-2) providing previouslyutilized attenuated vaccine strains with documented characteristics, andALVAC providing an example of a poxvirus whose replication is restrictedto avian species. Results from these in vivo analyses clearlydemonstrate the highly attenuated properties of NYVAC relative to thevaccinia virus strains, WR, WYETH and COPENHAGEN (VC-2) (Tables 14-20).Significantly, the LD₅₀ values for NYVAC were comparable to thoseobserved with the avian host restricted avipoxvirus, ALVAC. Deaths dueto NYVAC, as well as ALVAC, were observed only when extremely high dosesof virus were administered via the intracranial route (Example 11,Tables 14, 15, 19). It has not yet been established whether these deathswere due to nonspecific consequences of inoculation of a high proteinmass. Results from analyses in immunocompromised mouse models (nude andCY-treated) also demonstrate the relatively high attenuationcharacteristics of NYVAC, as compared to WR, WYETH and COPENHAGENstrains (Tables 17 and 18). Significantly, no evidence of disseminatedvaccinia infection or vaccinial disease was observed in NYVAC-inoculatedanimals or ALVAC-inoculated animals over the observation period. Thedeletion of multiple virulence-associated genes in NYVAC shows asynergistic effect with respect to pathogenicity. Another measure of theinocuity of NYVAC was provided by the intradermal administration onrabbit skin (Tables 17 and 18). Considering the results with ALVAC, avirus unable to replicate in nonavian species, the ability to replicateat the site of inoculation is not the sole correlate with reactivity,since intradermal inoculation of ALVAC caused areas of induration in adose dependent manner. Therefore, it is likely that factors other thanthe replicative capacity of the virus contribute to the formation of thelesions. Deletion of specific virulence-associated genes in NYVACprevents lesion occurrence.

Together, the results in this Example and in foregoing Examples,including Example 11, demonstrate the highly attenuated nature of NYVACrelative to WR, and the previously utilized vaccinia virus vaccinestrains, WYETH and COPENHAGEN. In fact, the pathogenic profile of NYVAC,in the animal model systems tested, was similar to that of ALVAC, apoxvirus known to productively replicate only in avian species. Theapparently restricted capacity of NYVAC to productively replicate oncells derived from humans (Table 16) and other species, including themouse, swine, dog and horse, provides a considerable barrier that limitsor prevents potential transmission to unvaccinated contacts or to thegeneral environment in addition to providing a vector with reducedprobability of dissemination within the vaccinated individual.

Significantly, NYVAC-based vaccine candidates have been shown to beefficacious. NYVAC recombinants expressing foreign gene products from anumber of pathogens have elicited immunological responses towards theforeign gene products in several animal species, including primates. Inparticular, a NYVAC-based recombinant expressing the rabies glycoproteinwas able to protect mice against a lethal rabies challenge. The potencyof the NYVAC-based rabies glycoprotein recombinant was comparable to thePD₅₀ value for a COPENHAGEN-based recombinant containing the rabiesglycoprotein in the tk locus (Table 20). NYVAC-based recombinants havealso been shown to elicit measles virus neutralizing antibodies inrabbits and protection against pseudorabies virus and Japaneseencephalitis virus challenge in swine. The highly attenuated NYVACstrain confers safety advantages with human and veterinary applications(Tartaglia et al., 1992). Furthermore, the use of NYVAC as a generallaboratory expression vector system may greatly reduce the biologicalhazards associated with using vaccinia virus.

By the following criteria, the results of this Example and the Examplesherein, including Example 11, show NYVAC to be highly attenuated: a) nodetectable induration or ulceration at site of inoculation (rabbitskin); b) rapid clearance of infectious virus from intradermal site ofinoculation (rabbit skin); c) absence of testicular inflammation (nudemice); d) greatly reduced virulence (intracranial challenge, boththree-week old and newborn mice); e) greatly reduced pathogenicity andfailure to disseminate in immunodeficient subjects (nude andcyclophosphamide treated mice); and f) dramatically reduced ability toreplicate on a variety of human tissue culture cells. Yet, in spite ofbeing highly attenuated, NYVAC, as a vector, retains the ability toinduce strong immune responses to extrinsic antigens.

TABLE 16 Replication of COPENHAGEN (VC-2), NYVAC and ALVAC in avian orhuman derived cell lines Hours post- Yield^(a) Cells infection VC-2NYVAC ALVAC % Yield CEF  0 3.8^(b) 3.7 4.5 24 8.3 7.8 6.6 48 8.6 7.9 7.772 8.3 7.7 7.5 25 72A^(c) <1.4 1.8 3.1 MRC-5  0 3.8 3.8 4.7 72 7.2 4.63.8 0.25 72A 2.2 2.2 3.7 WISH*  0 3.4 3.4 4.3 72 7.6 2.2 3.1 0.0004 72A—^(d) 1.9 2.9 DETROIT  0 3.8 3.7 4.4 72 7.2 5.4 3.4 1.6 72A 1.7 1.7 2.9HEL  0 3.8 3.5 4.3 72 7.5 4.6 3.3 0.125 72A 2.5 2.1 3.6 JT-1  0 3.1 3.14.1 72 6.5 3.1 4.2 0.039 72A 2.4 2.1 4.4 HNK  0 3.8 3.7 4.7 72 7.6 4.53.6 0.079 72A 3.1 2.7 3.7 ^(a)Yield of NYVAC at 72 hours post-infectionexpressed as a percentage of yield of VAC-2 after 72 hours on the samecell line. ^(b)Titer expressed as LOG₅₀ pfu per ml. ^(c)Sample wasincubated in the presence of 40 g/ml of cytosine arabinoside. ^(d)Notdetermined. *ATCC #CCL25 Human amnionic cells.

TABLE 17 Induration and ulceration at the site of intradermalinoculation of the rabbit skin VIRUS INDURATION ULCERATION STRAINDOSE^(a) Size^(b) Days^(c) Size Days WR 10⁴ 386 30 88 30 10⁵ 622 35 14932 10⁶ 1057 34 271 34 10⁷ 877 35 204 35 10⁸ 581 25 88 26 WYETH 10⁴ 32 5 —^(d) — 10⁵ 116 15 — — 10⁶ 267 17 3 15 10⁷ 202 17 3 24 10⁸ 240 29 12 31VC-2 10⁴ 64 7 — — 10⁵ 86 8 — — 10⁶ 136 17 — — 10⁷ 167 21 6 10 10⁸ 155 326 8 NYVAC 10⁴ — — — — 10⁵ — — — — 10⁶ — — — — 10⁷ — — — — 10⁸ — — — —^(a)pfu of indicated vaccinia virus in 0.1 ml PBS inoculatedintradermally into one site. ^(b)mean maximum size of lesions (mm²)^(c)mean time after inoculation for complete healing of lesion. ^(d)nolesions discernable.

TABLE 18 Persistence of poxviruses at the site of intradermalinoculation Total Virus Virus Inoculum Dose Recovered WR  8.0^(a) 6.147.0 6.26 6.0 6.21 WYETH 8.0 3.66 7.0 4.10 6.0 3.59 VC-2 8.0 4.47 7.04.74 6.0 3.97 NYVAC 8.0 0 7.0 0 6.0 0 ^(a)expressed as log₁₀ pfu.

TABLE 19 Virulence studies in immunocompromised mice LD₅₀ ^(a) PoxvirusCyclophosphamide Strain Nude mice treated mice WR 422  42 VC-2 >10⁹<1.65 × 10⁵  WYETH  1.58 × 10⁷ 1.83 × 10⁶ NY VAC >5.50 × 10⁸ 7.23 × 10⁸ALVAC >10⁹ ≧5.00 × 10^(8b)   ^(a)Calculated 50% lethal dose (pfu) fornude or cyclophosphamide treated mice by the indicated vaccinia virusesand for ALVAC by intraperitoneal route. ^(b)5 out of 10 mice died at thehighest dose of 5 × 10⁸ pfu.

TABLE 20 Comparative efficacy of NYVAC-RG and ALVAC-RG in miceRecombinant PD₅₀ ^(a) VV-RG 3.74 ALVAC-RG 3.86 NYVAC-RG 3.70 ^(a)Four tosix week old mice were inoculated in the footpad with 50-100 μl of arange of dilutions (2.0-8.0 log₁₀ tissue culture infection dose 50%(TCID₅₀) of either the VV-RG (Kieny et al., 1984), ALVAC-RG (vCP65) orNYVAC-RG (vP879). At day 14, mice of each group were challenged byintracranial inoculation of 30 μl of a live CVS strain rabies viruscorresponding to 15 lethal dose 50% (LD₅₀) per mouse. At day 28,surviving mice were counted and a protective dose 50% (PD₅₀) wascalculated.

Example 13 Construction of TROVAC Recombinants Expressing theHemagglutinin Glycoproteins of Avian Influenza Viruses

This Example describes the development of fowlpox virus recombinantsexpressing the hemagglutinin genes of three serotypes of avian influenzavirus.

Cells and Viruses. Plasmids containing cDNA clones of the H4, H5 and H7hemagglutinin genes were obtained from Dr. Robert Webster, St. JudeChildren's Research Hospital, Memphis, Tenn. The strain of FPVdesignated FP-1 has been described previously (Taylor et al., 1988a, b).It is a vaccine strain useful in vaccination of day old chickens. Theparental virus strain Duvette was obtained in France as a fowlpox scabfrom a chicken. The virus was attenuated by approximately 50 serialpassages in chicken embryonated eggs followed by 25 passages on chickembryo fibroblast (CEF) cells. This virus was obtained in September 1980by Rhone Merieux, Lyon, France, and a master viral seed established. Thevirus was received by Virogenetics in September 1989, where it wassubjected to four successive plaque purifications. One plaque isolatewas further amplified in primary CEF cells and a stock virus, designatedas TROVAC, was established. The stock virus used in the in vitrorecombination test to produce TROVAC-AIH5 (vFP89) and TROVAC-AIH4(vFP92) had been further amplified though 8 passages in primary CEFcells. The stock virus used to produce TROVAC-AIH7 (vFP100) had beenfurther amplified through 12 passages in primary CEF cells.

Construction of Fowlpox Insertion Plasmid at F8 Locus. Plasmid pRW731.15contains a 10 kbp PvuII-PvuII fragment cloned from TROVAC genomic DNA.The nucleotide sequence was determined on both strands for a 3659 bpPvuII-EcoRV fragment. This sequence is shown in FIG. 11 (SEQ ID NO:67).The limits of an open reading frame designated in this laboratory as F8were determined within this sequence. The open reading frame isinitiated at position 495 and terminates at position 1887. A deletionwas made from position 779 to position 1926, as described below.

Plasmid pRW761 is a sub-clone of pRW731.15 containing a 2430 bpEcoRV-EcoRV fragment. Plasmid pRW761 was completely digested with XbaIand partially digested with SspI. A 3700 bp XbaI-SspI band was isolatedand ligated with the annealed double-stranded oligonucleotides JCA017(SEQ ID NO:37) and JCA018 (SEQ ID NO:38).

JCA017 (SEQ ID NO: 37)5′ CTAGACACTTTATGTTTTTTAATATCCGGTCTTAAAAGCTTCCCGGG GATCCTTATACGGGGAATAAT3′ JCA018 (SEQ ID NO: 38)5′ ATTATTCCCCGTATAAGGATCCCCCGGGAAGCTTTTAAGACCGGATA TTAAAAAACATAAAGTGT 3′

The plasmid resulting from this ligation was designated pJCA002. PlasmidpJCA004 contains a non-pertinent gene linked to the vaccinia virus H6promoter in plasmid pJCA002. The sequence of the vaccinia virus H6promoter has been previously described (Taylor et al., 1988a, b; Guo etal. 1989; Perkus et al., 1989). Plasmid pJCA004 was digested with EcoRVand BamHI which deletes the non-pertinent gene and a portion of the 3′end of the H6 promoter. Annealed oligonucleotides RW178 (SEQ ID NO:48)and RW179 (SEQ ID NO:49) were cut with EcoRV and BamHI and insertedbetween the EcoRV and BamHI sites of JCA004 to form pRW846.

RW178 (SEQ ID NO: 48):5′ TCATTATCGCGATATCCGTGTTAACTAGCTAGCTAATTTTTATTCCC GGGATCCTTATCA 3′RW179 (SEQ ID NO: 49):5′ GTATAAGGATCCCGGGAATAAAAATTAGCTAGCTAGTTAACACGGAT ATCGCGATAATGA 3′Plasmid pRW846 therefore contains the H6 promoter 5′ of EcoRV in thede-ORFed F8 locus. The HincII site 3′ of the H6 promoter in pRW846 isfollowed by translation stop codons, a transcriptional stop sequencerecognized by vaccinia virus early promoters (Yuen et al., 1987) and aSmaI site.

Construction of Fowlpox Insertion Plasmid at F7 Locus. The original F7non-de-ORFed insertion plasmid, pRW731.13, contained a 5.5 kb FP genomicPvuII fragment in the PvuII site of pUC9. The insertion site was aunique HincII site within these sequences. The nucleotide sequence shownin FIG. 12 (SEQ ID NO:68) was determined for a 2356 bp regionencompassing the unique HincII site. Analysis of this sequence revealedthat the unique HincII site (FIG. 12, underlined) was situated within anORF encoding a polypeptide of 90 amino acids. The ORF begins with an ATGat position 1531 and terminates at position 898 (positions marked byarrows in FIG. 12).

The arms for the de-ORFed insertion plasmid were derived by PCR usingpRW731.13 as template. A 596 bp arm (designated as HB) corresponding tothe region upstream from the ORF was amplified with oligonucleotidesF73PH2 (SEQ ID NO:50) (5′-GACAATCTAAGTCCTATATTAGAC-3′) and F73PB (SEQ IDNO:51) (5′-GGATTTTTAGGTAGACAC-3′). A 270 bp arm (designated as EH)corresponding to the region downstream from the ORF was amplified usingoligonucleotides F75PE (SEQ ID NO:52) (5′-TCATCGTCTTCATCATCG-3′) andF73PH1 (SEQ ID NO:53) (5′-GTCTTAAACTTATTGTAAGGGTATACCTG-3′).

Fragment EH was digested with EcoRV to generate a 126 bp fragment. TheEcoRV site is at the 3′-end and the 5′-end was formed, by PCR, tocontain the 3′ end of a HincII site. This fragment was inserted intopBS-SK (Stratagene, La Jolla, Calif.) digested with HincII to formplasmid pF7D1. The sequence was confirmed by dideoxynucleotide sequenceanalysis. The plasmid pF7D1 was linearized with ApaI, blunt-ended usingT4 DNA polymerase, and ligated to the 596 bp HB fragment. The resultantplasmid was designated as pF7D2. The entire sequence and orientationwere confirmed by nucleotide sequence analysis.

The plasmid pF7D2 was digested with EcoRV and BglII to generate a 600 bpfragment. This fragment was inserted into pBS-SK that was digested withApaI, blunt-ended with T4 DNA polymerase, and subsequently digested withBamHI. The resultant plasmid was designated as pF7D3. This plasmidcontains an HB arm of 404 bp and a EH arm of 126 bp.

The plasmid pF7D3 was linearized with XhoI and blunt-ended with theKlenow fragment of the E. coli DNA polymerase in the presence of 2 mMdNTPs. This linearized plasmid was ligated with annealedoligonucleotides F7MCSB (SEQ ID NO:54)(5′-AACGATTAGTTAGTTACTAAAAGCTTGCTGCAGCCCGGGTTTTTTATTAGTTTAGTT AGTC-3′)and F7MCSA (SEQ ID NO:55) (5′-GACTAACTAACTAATAAAAAACCCGGGCTGCAGCAAGCTTTTTGTAACTAACTAATCGTT-3′). This was performed toinsert a multiple cloning region containing the restriction sites forHindIII, PstI and SmaI between the EH and HB arms. The resultant plasmidwas designated as pF7DO.

Construction of Insertion Plasmid for the H4 Hemagglutinin at the F8Locus. A cDNA copy encoding the avian influenza H4 derived fromA/Ty/Min/833/80 was obtained from Dr. R. Webster in plasmid pTM4H833.The plasmid was digested with HindIII and NruI and blunt-ended using theKlenow fragment of DNA polymerase in the presence of dNTPs. Theblunt-ended 2.5 kbp HindIII-NruI fragment containing the H4 codingregion was inserted into the HincII site of pIBI25 (InternationalBiotechnologies, Inc., New Haven, Conn.). The resulting plasmid pRW828was partially cut with BanII, the linear product isolated and recut withHindIII. Plasmid pRW828 now with a 100 bp HindIII-BanII deletion wasused as a vector for the synthetic oligonucleotides RW152 (SEQ ID NO:56)and RW153 (SEQ ID NO:57). These oligonucleotides represent the 3′portion of the H6 promoter from the EcoRV site and align the ATG of thepromoter with the ATG of the H4 cDNA.

RW152 (SEQ ID NO: 56):5′ GCACGGAACAAAGCTTATCGCGATATCCGTTAAGTTTGTATCGTAATGCTATCAATCACGATTCTGTTCCTGCTCATAGCAGAGGGCTCATCTCAGA AT 3′ RW153 (SEQ IDNO: 57): 5′ ATTCTGAGATGAGCCCTCTGCTATGAGCAGGAACAGAATCGTGATTGATAGCATTACGATACAAACTTAACGGATATCGCGATAAGCTTTGTTCCGT GC 3′

The oligonucleotides were annealed, cut with BanII and HindIII andinserted into the HindIII-BanII deleted pRW828 vector described above.The resulting plasmid pRW844 was cut with EcoRV and DraI and the 1.7 kbpfragment containing the 3′ H6 promoted H4 coding sequence was insertedbetween the EcoRV and HincII sites of pRW846 (described previously)forming plasmid pRW848. Plasmid pRW848 therefore contains the H4 codingsequence linked to the vaccinia virus H6 promoter in the de-ORFed F8locus of fowlpox virus.

Construction of Insertion Plasmid for H5 Hemagglutinin at the F8 Locus.A cDNA clone of avian influenza H5 derived from A/Turkey/Ireland/1378/83was received in plasmid pTH29 from Dr. R. Webster. Syntheticoligonucleotides RW10 (SEQ ID NO:58) through RW13 (SEQ ID NO:61) weredesigned to overlap the translation initiation codon of the previouslydescribed vaccinia virus H6 promoter with the ATG of the H5 gene. Thesequence continues through the 5′ SalI site of the H5 gene and beginsagain at the 3′ H5 DraI site containing the H5 stop codon.

RW10 (SEQ ID NO: 58): 5′ GAAAAATTTAAAGTCGACCTGTTTTGTTGAGTTGTTTGCGTGGTAACCAATGCAAATCTGGTCACT 3′ RW11 (SEQ ID NO: 59):5′ TCTAGCAAGACTGACTATTGCAAAAAGAAGCACTATTTCCTCCATTA CGATACAAACTTAACGGAT3′ RW12 (SEQ ID NO: 60):5′ ATCCGTTAAGTTTGTATCGTAATGGAGGAAATAGTGCTTCTTTTTGCAATAGTCAGTCTTGCTAGAAGTGACCAGATTTGCATTGGT 3′ RW13 (SEQ ID NO:61):5′ TACCACGCAAACAACTCAACAAAACAGGTCGACTTTAAATTTTTCTG CA 3′

The oligonucleotides were annealed at 95° C. for three minutes followedby slow cooling at room temperature. This results in the followingdouble strand structure with the indicated ends.

Cloning of oligonucleotides between the EcoRV and PstI sites of pRW742Bresulted in pRW744. Plasmid pRW742B contains the vaccinia virus H6promoter linked to a non-pertinent gene inserted at the HincII site ofpRW731.15 described previously. Digestion with PstI and EcoRV eliminatesthe non-pertinent gene and the 3′-end of the H6 promoter. Plasmid pRW744now contains the 3′ portion of the H6 promoter overlapping the ATG ofavian influenza H5. The plasmid also contains the H5 sequence throughthe 5′ SalI site and the 3′ sequence from the H5 stop codon (containinga DraI site). Use of the DraI site removes the H5 3′ non-coding end. Theoligonucleotides add a transcription termination signal recognized byearly vaccinia virus RNA polymerase (Yuen et al., 1987). To complete theH6 promoted H5 construct, the H5 coding region was isolated as a 1.6 kpbSalI-DraI fragment from pTH29. Plasmid pRW744 was partially digestedwith DraI, the linear fragment isolated, recut with SalI and the plasmidnow with eight bases deleted between SalI and DraI was used as a vectorfor the 1.6 kpb pTH29 SalI and DraI fragment. The resulting plasmidpRW759 was cut with EcoRV and DraI. The 1.7 kbp PRW759 EcoRV-DraIfragment containing the 3′ H6 promoter and the H5 gene was insertedbetween the EcoRV and HincII sites of pRW846 (previously described). Theresulting plasmid pRW849 contains the H6 promoted avian influenza virusH5 gene in the de-ORFed F8 locus.

Construction of Insertion Vector for H7 Hemagglutinin at the F7 Locus.Plasmid pCVH71 containing the H7 hemagglutinin from A/CK/VIC/1/85 wasreceived from Dr. R. Webster. An EcoRI-BamHI fragment containing the H7gene was blunt-ended with the Klenow fragment of DNA polymerase andinserted into the HincII site of pIBI25 as PRW827. Syntheticoligonucleotides RW165 (SEQ ID NO:62) and RW166 (SEQ ID NO:63) wereannealed, cut with HincII and StyI and inserted between the EcoRV andStyI sites of pRW827 to generate pRW845.

RW165 (SEQ ID NO: 62):5′ GTACAGGTCGACAAGCTTCCCGGGTATCGCGATATCCGTTAAGTTTGTATCGTAATGAATACTCAAATTCTAATACTCACTCTTGTGGCAGCCATTCACACAAATGCAGACAAAATCTGCCTTGGACATCAT 3′ RW166 (SEQ ID NO: 63):5′ ATGATGTCCAAGGCAGATTTTGTCTGCATTTGTGTGAATGGCTGCCACAAGAGTGAGTATTAGAATTTGAGTATTCATTACGATACAAACTTAACGGATATCGCGATACCCGGGAAGCTTGTCGACCTGTAC 3′

Oligonucleotides RW165 (SEQ ID NO:62) and RW166 (SEQ ID NO:63) link the3′ portion of the H6 promoter to the H7 gene. The 3′ non-coding end ofthe H7 gene was removed by isolating the linear product of an ApaLIdigestion of pRW845, recutting it with EcoRI, isolating the largestfragment and annealing with synthetic oligonucleotides RW227 (SEQ IDNO:64) and RW228 (SEQ ID NO:65). The resulting plasmid was pRW854.

RW227 (SEQ ID NO: 64):5′ ATAACATGCGGTGCACCATTTGTATATAAGTTAACGAATTCCAAGTC AAGC 3′ RW228 (SEQ IDNO: 65): 5′ GCTTGACTTGGAATTCGTTAACTTATATACAAATGGTGCACCGCATG TTAT 3′The stop codon of H7 in PRW854 is followed by an HpaI site. Theintermediate H6 promoted H7 construct in the de-ORFed F7 locus(described below) was generated by moving the pRW854 EcoRV-HpaI fragmentinto pRW858 which had been cut with EcoRV and blunt-ended at its PstIsite. Plasmid pRW858 (described below) contains the H6 promoter in an F7de-ORFed insertion plasmid.

The plasmid pRW858 was constructed by insertion of an 850 bp SmaI/HpaIfragment, containing the H6 promoter linked to a non-pertinent gene,into the SmaI site of pF7DO described previously. The non-pertinentsequences were excised by digestion of pRW858 with EcoRV (site 24 bpupstream of the 3′-end of the H6 promoter) and PstI. The 3.5 kbresultant fragment was isolated and blunt-ended using the Klenowfragment of the E. coli DNA polymerase in the presence of 2 mM dNTPs.This blunt-ended fragment was ligated to a 1700 bp EcoRV/HpaI fragmentderived from pRW854 (described previously). This EcoRV/HpaI fragmentcontains the entire AIV HA (H7) gene juxtaposed 3′ to the 3′-most 24 bpof the VV H6 promoter. The resultant plasmid was designated pRW861.

The 126 bp EH arm (defined previously) was lengthened in pRW861 toincrease the recombination frequency with genomic TROVAC DNA. Toaccomplish this, a 575 bp AccI/SnaBI fragment was derived from pRW731.13 (defined previously). The fragment was isolated and insertedbetween the AccI and NaeI sites of pRW861. The resultant plasmid,containing an EH arm of 725 bp and a HB arm of 404 bp flanking the AIVH7 gene, was designated as pRW869. Plasmid pRW869 therefore consists ofthe H7 coding sequence linked at its 5′ end to the vaccinia virus H6promoter. The left flanking arm consists of 404 bp of TROVAC sequenceand the right flanking arm of 725 bp of TROVAC sequence which directsinsertion to the de-ORFed F7 locus.

Development of TROVAC-Avian Influenza Virus Recombinants. Insertionplasmids containing the avian influenza virus HA coding sequences wereindividually transfected into TROVAC infected primary CEF cells by usingthe calcium phosphate precipitation method previously described(Panicali et al., 1982; Piccini et al., 1987). Positive plaques wereselected on the basis of hybridization to HA specific radiolabelledprobes and subjected to sequential rounds of plaque purification until apure population was achieved. One representative plaque was thenamplified to produce a stock virus. Plasmid pRW849 was used in an invitro recombination test to produce recombinant TROVAC-AIH5 (vFP89)expressing the H5 hemagglutinin. Plasmid pRW848 was used to producerecombinant TROVAC-AIH4 (vFP92) expressing the H4 hemagglutinin. PlasmidpRW869 was used to produce recombinant TROVAC-AIH7 (vFP100) expressingthe H7 hemagglutinin.

Immunofluorescence. In influenza virus infected cells, the HA moleculeis synthesized and glycosylated as a precursor molecule at the roughendoplasmic reticulum. During passage to the plasma membrane itundergoes extensive post-translational modification culminating inproteolytic cleavage into the disulphide linked HA₁ and HA₂ subunits andinsertion into the host cell membrane where it is subsequentlyincorporated into mature viral envelopes. To determine whether the HAmolecules produced in cells infected with the TROVAC-AIV recombinantviruses were expressed on the cell surface, immunofluorescence studieswere performed. Indirect immunofluorescence was performed as described(Taylor et al., 1990). Surface expression of the H5 hemagglutinin inTROVAC-AIH5, H4 hemagglutinin in TROVAC-AIH4 and H7 hemagglutinin inTROVAC-AIH7 was confirmed by indirect immunofluorescence. Expression ofthe H5 hemagglutinin was detected using a pool of monoclonal antibodiesspecific for the H5HA. Expression of the H4HA was analyzed using a goatmonospecific anti-H4 serum. Expression of the H7HA was analyzed using aH7 specific monoclonal antibody preparation.

Immunoprecipitation. It has been determined that the sequence at andaround the cleavage site of the hemagglutinin molecule plays animportant role in determining viral virulence since cleavage of thehemagglutinin polypeptide is necessary for virus particles to beinfectious. The hemagglutinin proteins of the virulent H5 and H7 virusespossess more than one basic amino acid at the carboxy terminus of HA1.It is thought that this allows cellular proteases which recognize aseries of basic amino acids to cleave the hemagglutinin and allow theinfectious virus to spread both in vitro and in vivo. The hemagglutininmolecules of H4 avirulent strains are not cleaved in tissue cultureunless exogenous trypsin is added.

In order to determine that the hemagglutinin molecules expressed by theTROVAC recombinants were authentically processed, immunoprecipitationexperiments were performed as described (Taylor et al., 1990) using thespecific reagents described above.

Immunoprecipitation analysis of the H5 hemagglutinin expressed byTROVAC-AIH5 (vFP89) showed that the glycoprotein is evident as the twocleavage products HA₁ and HA₂ with approximate molecular weights of 44and 23 kDa, respectively. No such proteins were precipitated fromuninfected cells or cells infected with parental TROVAC. Similarlyimmunoprecipitation analysis of the hemagglutinin expressed byTROVAC-AIH7 (vFP100) showed specific precipitation of the HA₂ cleavageproduct. The HA₁ cleavage product was not recognized. No proteins werespecifically precipitated from uninfected CEF cells or TROVAC infectedCEF cells. In contrast, immunoprecipitation analysis of the expressionproduct of TROVAC-AIH4 (vFP92) showed expression of only the precursorprotein HA₀. This is in agreement with the lack of cleavage of thehemagglutinins of avirulent subtypes in tissue culture. No H4 specificproteins were detected in uninfected CEF cells or cells infected withTROVAC. Generation of recombinant virus by recombination, in situhybridization of nitrocellulose filters and screening forB-galactosidase activity are as previously described (Panicali et al.,1982; Perkus et al., 1989).

Example 14 Generation of an ALVAC Recombinant Expressing HIV1 gag (+pro)(IIIB), gp120 (MN) (+transmembrane) Epitopes

A plasmid, pHXB2D, containing HIV1 (IIIB) cDNA sequence (Ratner et al,1985), was obtained from Robert Gallo (NCI, NIH). The sequence encodingthe 5′-end of the gag gene was cloned between vaccinia virus tk flankingarms. This was accomplished by cloning the 1,625 bp BglII fragment ofpHXB2D, containing the 5′-end of the gag gene, into the 4,075 bp BglIIfragment of pSD542VCVQ. The plasmid generated by this manipulation iscalled pHIVG2.

The 3′-end of the gag gene was then cloned into pHIVG2. This wasaccomplished by cloning a 280 bp ApaI-BamHI PCR fragment, containing the3′-end of the gag gene, into the 5,620 bp ApaI-BamHI fragment of pHIVG2.(This PCR fragment was generated from the plasmid, pHXB2D, with theoligonucleotide primers, HIVP5 (SEQ ID NO:69; 5′-TGTGGCAAAGAAGGGC-3′)and HIVP6 (SEQ ID NO:70; 5′-TTGGATCCTTATTGTGACGAGGGGTC-3′).) The plasmidgenerated by this manipulation is called pHIVG3.

The I3L promoter was then cloned upstream of the gag gene. This wasaccomplished by cloning the oligonucleotides, HIVL17 (SEQ ID NO:71;5′-GATCTTGAGATAAAGTGAAAATATATATCATTATATTACAAAGTACAATTATTTAGGTTTAATCATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGAT-3′) and HIVL18 (SEQID NO:72; 5′-CGATCTAATTCTCCCCCGCTTAATACTGACGCTCTCGCACCCATGATTAAACCTAAATAATTGTACTTTGTAATATAATGATATATATTTTCACTTTATCTCAA-3′), encoding the vaccinia virus I3L promoter and the 5′-end ofthe gag gene, into the 5,540 bp partial BglII-ClaI fragment of pHIVG3.The plasmid generated by this manipulation is called pHIVG4.

The portion of the gag gene encoding p24, p2, p7 and p6 was theneliminated. This was accomplished by cloning the oligonucleotides,HIVL19 (SEQ ID NO:73; 5′-CTGACACAGGACACAGCAATCAGGTCAGCCAAAATTACTAATTTTTATCTCGAGGTCGACAGGACCCG-3′) andHIVL20 (SEQ ID NO:74; 5′-GATCCGGGTCCTGTCGACCTCGAGATAAAAATTAGTAATTTTGGCTGACCTGATTGCTGTGTCCTGTGTCAG-3′), into the 4,450 bp partialPvuII-BamHI fragment of pHIVG4. The plasmid generated by thismanipulation is called pHIVG5.

The remainder of the gag gene, as well as the pol gene, was then cloneddownstream of the p17 “gene”. This was accomplished by cloning the 4,955bp ClaI-SalI fragment of pHXB2D, containing most of the gag gene and allof the pol gene, into the 4,150 bp ClaI-SalI fragment of pHIVG5. Theplasmid generated by this manipulation is called pHIVG6.

Extraneous 3′-noncoding sequence was then eliminated. This wasaccomplished by cloning a 360 bp AflII-BamHI PCR fragment, containingthe 3′-end of the pol gene, into the 8,030 bp AflII-BamHI fragment ofpHIVG6. (This PCR fragment was generated from the plasmid, pHXB2D, withthe oligonucleotide primers, HIVP7 (SEQ ID NO:75; 5′-AAGAAAATTATAGGAC-3′) and HIVP8 (SEQ ID NO:76; 5′-TTGGATCCCTAATCCTCATCCTGT-3′).) The plasmid generated by this manipulation iscalled pHIVG7.

The I3L-promoted gag and pol genes were then cloned between canary poxC3 flanking arms. This was accomplished by cloning the 4,360 bp partialBglII-BamHI fragment of pHIVG7, containing the I3L-promoted gag and polgenes, into the BamHI site of pVQH6CP3L. The plasmid generated by thismanipulation is called pHIVGE14.

The H6-promoted HIV1gp120(MN) (+transmembrane) “gene” (Gurgo et al,1988) was then cloned into pHIVGE14. This was accomplished by cloningthe 1,700 bp NruI-SmaI fragment of pC5HIVMN120T, containing thegp120(+transmembrane) “gene”, into the 11,400 bp NruI-SmaI fragment ofpHIVGE14. The plasmid generated by this manipulation is calledpHIVGE14T.

Most of the pol gene was then removed. This was accomplished by cloninga 540 bp ApaI-BamHI PCR fragment, containing the 3′-end of the HIV1protease “gene”, into the 10,000 bp ApaI-BamHI fragment of pHIVGE14T.(This PCR fragment was generated from the plasmid, pHIVG7, with theoligonucleotide primers, HIVP5 and HIVP37 (SEQ ID NO:77;5′-AAAGGATCCCCCGGGTTAAAAATTTAAAGTGCAACC-3′).) This manipulation removesmost of the pol gene, but leaves the protease “gene” intact. The plasmidgenerated by this manipulation is called pHIV32. The DNA sequence ofpHIV32 (SEQ ID NOS: 78 and 79) is shown in FIGS. 14A-14C which shows thenucleotide sequence of the H6-promoted HIV1gp120(+transmembrane) geneand the I3L-promoted HIV1gag(+pro) gene contained in pHIV32:

gag (+pro) and gp120 (+transmembrane) FEATURES From To/Span Descriptionfrag 1 56 C3 flanking arm frag 162 76 (C) HIV1 (IIIB) env transmembraneregion frag 1728 163 (C) HIV1 (MM) gp120 gene frag 1853 1729 (C)vaccinia H6 promoter frag 1925 1983 vaccinia I3L promoter frag 1984 3746HIV1 (IIB) gag/pro gene frag 3753 3808 C3 flanking arm

The DNA sequence of the ALVAC C3 flanking arm (SEQ ID NOS: 80 and 81) isshown in FIGS. 15A-F. FIGS. 15A to 15F show the nucleotide sequence ofthe C3 locus in pVQH6CP3L:

C3 LOCUS pVQH6CP3L FEATURES From To/Span Description frag 1 1460 C3flanking arm frag 1461 1501 Cloning sites frag 1630 1502 H6 promoterfrag 1717 4291 C3 flanking arm

pHIV32 was used in vitro recombination with ALVAC as the rescuing virusto yield vCP205.

vCP205 (ALVAC-MN120TMG) was deposited on Mar. 6, 1997 under the terms ofthe Budapest Treaty with the American Type Culture Collection (ATCC),P.O. Box 1549, Manassas, Va. 20108 USA under ATCC accession numberVR-2557.

Example 15 Generation of an ALVAC Recombinant Expressing HIV1 gag (+pro)(IIIB), gp120 (MN) (+transmembrane) and 2 nef (BRU) Epitopes

Expression cassettes encoding two nef CTL epitopes, CTL1 (amino acids66-147) and CTL2 (amino acids 182-206) (Wain-Hobson et al, 1985; Nixonand McMichael, 1991), were then inserted into vCP205. The insertionplasmid, p2-60-HIV.3, containing the nef CTL epitopes, was generated bythe following procedure. The I3L-promoted CTL2 epitope was cloned intopBSH6. This was accomplished by cloning a 255 bp PCR HindIII-XhoIfragment, containing the I3L-promoted CTL2 epitope, into the 3,100 bpHindIII-XhoI fragment of pBSH6. (The 255 bp PCR fragment was generatedby the following procedure. A 216 bp PCR fragment, containing theI3L-promoter and the 5′-end of the CTL2 epitope, was generated from theplasmid, pMPI3H, with the oligonucleotide primers, VQPCRI3 (SEQ ID NO:82; 5′-ATCATCAAGCTTAATTAATTAGTTATTAGACAAGGTGAAAACGAAACTATTTGTAGCTTAATTATTAGACATCATGCAGTGGTTAAAC-3′) and I3PCRCTL (SEQ ID NO: 83;5′-CTAGCTACGTGATGAAATGCTAATCTAGAATCAAATCTCCACTCCATGATTAAACCTAAATAATTGTAC-3′). This 216 bp PCR fragment was then used as a template ina second PCR reaction with the oligonucleotide primers, VQPCRI3 andCTLPCR (SEQ ID NO: 84;5′-GAATTCCTCGAGGATCCTCTAGATTAACAATTTTTAAAATATTCAGGATGTAATTCTCTAGCTACGTGATGAAATGC-3′), to generate the PCR fragment, containing theI3L-promoted CTL2 epitope, that was digested with HindIII and XhoI andcloned into pBSH6). The plasmid generated by this manipulation is calledp2-60-HIV.1.

The H6-promoted CTL1 epitope was then cloned into p2-60-HIV.1. This wasaccomplished by cloning a 290 bp NruI-EcoRI fragment, containing theH6-promoted CTL1 epitope, into the 3,300 bp NruI-EcoRI fragment ofp2-60-HIV.1. (The 290 bp NruI-EcoRI fragment was generated by thefollowing procedure. A 195 bp PCR fragment, containing the H6-promoterand the 5′-end of the CTL1 epitope, was generated from the plasmid,pH6T2, with the oligonucleotide primers, H6PCR1 (SEQ ID NO: 85;5′-ACTACTAAGCTTCTTTATTCTATACTTAAAAAGTG-3′) and NCCPCR1 (SEQ ID NO: 86;5′-CAGCTGCTTTGTAAGTCATTGGTCTTAAAGGTACTTGAGGTGTTACTGGAAAACCTACCATTACGATACAAACTTAACGGATATCGCG-3′). This 195 bp PCR fragment and theoligonucleotides, NCC174A (SEQ ID NO: 87;5′-ACTTACAAAGCAGCTGTAGATCTTTCTCACTTTTTAAAAGAAAAAGGAGGTTTAGAAGGGCTAATTCATTCTCAACGAAGACAAGATATTCTTGATTTGTGG-3′) and NCC174B (SEQ ID NO:88; 5′-CCACAAATCAAGAATATCTTGTCTTCGTTGAGAATGAATTAGCCCTTCTAAACCTCCTTTTTCTTTTAAAAAGTGAGAAAGATCTACAGCTGCTTTGTAAGT-3′), were then used astemplates in a second PCR reaction with the oligonucleotide primers,H6PCR1 and NCCPCR2 (SEQ ID NO: 89;5′-CTGCCAATCAGGAAAATATCCTTGTGTATGATAAATCCACAAATCAAGAATATC-3′). Theresulting 317 bp PCR fragment, containing the H6-promoter and most ofthe CTL1 epitope, and the oligonucleotides, NCC291A (SEQ ID NO: 90;5′-GGATATTTTCCTGATTGGCAGAATTACACACCAGGACCAGGAGTCAGATACCCATTAACCTTTGGTTGGTGCTACAAGC-3′) and NCC291B (SEQ ID NO: 91;5′-GCTTGTAGCACCAACCAAAGGTTAATGGGTATCTGACTCCTGGTCCTGGTGTGTAATTCTGCCAATCAGGAAAATATCC-3′), were then used as templates in a third PCRreaction with the oligonucleotide primers, H6PCR1 and NCCPCR3 (SEQ IDNO: 92; 5′-ACTACTGAATTCTCGAGAAAAATTATGGTACTAGCTTGTAGCACCAACC-3′), togenerate the PCR fragment, containing the H6-promoted CTL1 epitope, thatwas digested with NruI and EcoRI and cloned into p2-60-HIV.1) Theplasmid generated by this manipulation is called p2-60-HIV.2.

The I3L-promoted CTL2 and H6-promoted CTL1 epitopes were then clonedbetween canarypox C6 flanking arms. This was accomplished by cloning the640 bp XhoI fragment of p2-60-HIV.2, containing the two (2) nef CTLepitopes, into the XhoI site of pC6L. The plasmid generated by thismanipulation is called p2-60-HIV.3. The DNA sequence of p2-60-HIV.3 (SEQID NOS: 93-96) is shown in FIG. 16. FIG. 16 shows the nucleotidesequence of the I3L-promoted nef CTL2 epitope and H6-promoted nef CTL1epitope contained in p2-60-HIV.3:

NEF CTL epitopes FEATURES From To/Span Description frag 1 51 C6 Left Armpept 175 98 (C) nef CTL2 frag 275 176 (C) I3L promoter frag 337 460 H6promoter pept 461 709 1 nef CTL1 frag 751 801 C6 Right Arm

The DNA sequence of the ALVAC C6 flanking arm (SEQ ID NOS: 97 and 98) isshown in FIGS. 17A-C. FIG. 17A to 17C show the nucleotide sequence ofthe C6 locus in pC6L:

C6 LOCUS pC6L FEATURES From To/Span Description frag 1 381 C6 flankingarm frag 382 447 Cloning sites frag 448 1615 C6 flanking arm

p2-60HIV.3 was used in in vitro recombination experiments with vCP205 asthe rescuing virus to yield vCP264.

Example 16 Generation of an ALVAC Recombinant Expressing HIV1 gag (+pro)(IIIB), gp120 (MN) (+transmembrane) and 2 nef (BRU) and 3 pol (IIIB) CTLEpitope Containing Regions

Expression cassettes encoding three (3) pol CTL epitopes, poll (aminoacids 172-219), pol2 (amino acids 325-383) and pol3 (amino acids461-519) (Ratner et al, 1985; Nixon and McMichael, 1991), were theninserted into vCP264. The insertion plasmid, pC5POLT5A, containing thethree (3) pol CTL epitopes, was generated by cloning a 948 bp XhoI-BamHIfragment, containing the H6-promoted poll epitope, the I3L-promoted pol2epitope and the 42K-promoted pol3 epitope, into the 2,940 bp XhoI-BamHIfragment of PBSK⁺. (The 948 bp XhoI-BamHI fragment was generated by thefollowing procedure. A 183 bp PCR fragment, containing the poll epitope,was generated from the plasmid, pHXB2D, with the oligonucleotideprimers, P1A (SEQ ID NO: 99;5′-TTTGTATCGTAATGATTGAGACTGTACCAGTAAAATTAAAGCC-3′) and P1B (SEQ ID NO:100; 5′-GGGCTGCAGGAATTCTAATCAATTAAGGCCCAATTTTTGAAATTTTCCCTTCCTTTTCCATCTCTG-3′). A 224 bp PCR fragment, containing the pol2 epitope, wasgenerated from the plasmid, pHXB2D, with the oligonucleotide primers,P2A (SEQ ID NO: 101;5′-ACAAAGTACAATTATTTAGGTTTAATCATGGCAATATTCCAAAGTAGCATGAC-3′) and P2B(SEQ ID NO: 102; 5′-ATCATCCTCGAGAAAAATTAGGTAAGTCCCCACCTCAACAGATG-3′). A236 bp PCR fragment, containing the pol3 epitope, was generated from theplasmid, pHXB2D, with the oligonucleotide primers, P3A (SEQ ID NO: 103;5′-AAAATATATAATTACAATATAAAATGCCACTAACAGAAGAAGCAGAGCTAGAACTGGC-3′) andP3B (SEQ ID NO: 104;5′-ATCATCTCTAGACTCGAGGATCCATAAAAATTATCCTGTTTTCAGATTTTTAAATGGCT C-3′). A340 bp PCR fragment, containing the I3L and H6 promoters (in ahead-to-head configuration) was generated from the plasmid, p2-60-HIV.2,with the oligonucleotide primers, P2IVH (SEQ ID NO: 105;5′-GTCATGCTACTTTTGAATATTGCCATGATTAAACCTAAATAATTGTACTTTG-3,) and IVHP1(SEQ ID NO: 106;5′-TTTAATTTTACTGGTACAGTCTCAATCATTACGATACAAACTTAACGGATATCGCG-3′). A 168bp PCR fragment, containing the 42K promoter, was generated from theplasmid, pVQ42KTh4.1, with the oligonucleotide primers, EPS42K (SEQ IDNO: 107; 5′-AATTGATTAGAATTCCTGCAGCCCGGGTCAAAAAAATATAAATG-3′) and 42 KP3B(SEQ ID NO: 108;5′-CCAGTTCTAGCTCTGCTTCTTCTGTTAGTGGCATTTTATATTGTAATTATATATTTTC-3′). A 511bp PCR fragment, containing the H6 promoter and I3L-promoted pol2epitope, was generated by using the 224 bp PCR fragment, containing thepol2 epitope, and the 340 bp PCR fragment, containing the I3L and H6promoters, as templates in a PCR reaction with the oligonucleotideprimers, P2B and IVHP1. A 347 bp PCR fragment, containing the42K-promoted pol3 epitope, was generated by using the 168 bp PCRfragment, containing the 42K promoter, and the 236 bp PCR fragment,containing the pol3 epitope, as templates in a PCR reaction with theoligonucleotide primers, IPS42K and P3B. A 506 bp PCR fragment,containing the poll epitope and the 42K-promoted pol3 epitope, wasgenerated by using the 183 bp PCR fragment, containing the poll epitope,and the 347 bp PCR fragment, containing the 42K-promoted pol3 epitope,as templates in a PCR reaction with the oligonucleotide primers, P1A andP3B. A 977 bp PCR fragment, containing the H6-promoted poll epitope, theI3L-promoted pol2 epitope and the 42K-promoted pol3 epitope, wasgenerated by using the 511 bp PCR fragment, containing the H6 promoterand I3L-promoted pol2 epitope, and the 506 bp PCR fragment, containingthe poll epitope and 42K-promoted pol3 epitope, as templates in a PCRreaction with the oligonucleotide primers, P2B and P3B. The 977 bp PCRfragment was then digested with XhoI and BamHI and cloned into the 2,940bp XhoI-BamHI fragment of pBSK⁺.) The plasmid generated by thismanipulation is called pBSPOLT5.

Nucleotide sequence analysis of pBSPOLT5 indicated that there was anerror in the pol2 epitope. In order to correct this mistake, the 948 bpXhoI-BamHI fragment, containing the H6-promoted poll epitope, theI3L-promoted pol2 epitope and the 42K-promoted pol3 epitope, was used asa template in a PCR reaction with the oligonucleotide primers, 13PCR1(SEQ ID NO: 109; 5′-ATCATCGGATCCAAGCTTACATCATGCAGTGG-3′) and FIXPOL2(SEQ ID NO: 110; 5′-ATCATCCTCGAGCTATTCAATTAGGTTGTAAGTCCCCACCTCAAC-3′).The resulting PCR fragment, containing the corrected I3L-promoted pol2epitope, was digested with HindIII and XhoI and cloned into the 3,650 bpHindIII-XhoI fragment of pBSPOLT5. The plasmid generated by thismanipulation is called pBSPOLT5A.

The H6-promoted poll epitope, I3L-promoted pol2 epitope and 42K-promotedpol3 epitope was then cloned between canary pox C5 flanking arms. Thiswas accomplished by cloning the 897 bp BamHI-XhoI fragment of pBSPOLT5A,containing the H6-promoted poll epitope, the I3L-promoted pol2 epitopeand the 42K-promoted pol3 epitope, into the 4,675 bp BamHI-XhoI fragmentof pNC5L-SP5. The plasmid generated by this manipulation is calledpC5POLT5A. The DNA sequence of pC5POLT5A (SEQ ID NOS: 111-115) is shownin FIGS. 18A-B. FIGS. 18A to 18B shows the nucleotide sequence of theI3L-promoted pol2 epitope, H6-promoted poll epitope and 42K-promotedpol3 epitope contained in pC5POLT5a:

POL CTL epitopes FEATURES From To/Span Description frag 1 50 C5 Left Armpept 272 92 (C) POL 2 frag 372 273 (C) I3L promoter frag 377 500 H6promoter pept 501 647 1 POL1 frag 676 782 _42K promoter pept 783 962 1POL 3 frag 986 1035 C5 Right Arm

The DNA sequence of the ALVAC C5 flanking arm (SEQ ID NOS: 116 and 117)is shown in FIGS. 19A-C. FIGS. 19A to 19C show the nucleotide sequenceof the C5 locus in pNC5L-SP5:

C5 LOCUS pNC5L-SP5 FEATURES From To/Span Description frag 1 1549 C5flanking arm frag 1550 1637 Cloning sites frag 1638 2049 C5 flanking arm

pC5POLT5A was used in in vitro recombination experiments with vCP264 asthe rescuing virus to yield vCP300.

Example 17 Restriction and Immunoprecipitation Analyses

Restriction enzyme analysis was performed to confirm that the HIV1sequences in vCP300 are in the proper loci. ALVAC, vCP205, vCP264 andvCP300 DNA were digested with HindIII, PstI or XhoI and the resultantfragments fractionated on an agarose gel. When the sizes of theresulting fragments were compared, it was determined that, as expected,the gag(+pro) and gp120(+transmembrane) genes were inserted into the C3locus, the nef epitopes were inserted into the C6 locus and the polepitopes were inserted into the C5 locus.

Immunoprecipitation analysis was performed to determine whether vCP300expresses authentic HIV1gag and gp120(+transmembrane) gene products.HeLa cell monolayers were either mock infected or infected at an m.o.i.of 10 pfu/cell with ALVAC or vCP300. Following an hour adsorptionperiod, the inoculum was aspirated and the cells were overlayed with 2mls of modified Eagle's medium (minus methionine) containing 2% fetalbovine serum and [³⁵S]-methionine (20 μCi/ml). Cells were harvested at18 hrs post-infection by the addition of 1 ml 3× buffer A (3% NP-40, 30mM Tris (pH7.4), 3 mM EDTA, 0.03% Na Azide and 0.6 mg/ml PMSF) and 50 ulaprotinin, with subsequent scraping of the cell monolayers. Lysates fromthe infected cells were analyzed for HIV1gag and gp120(+transmembrane)gene expression using serum from HIV1-seropositive individuals (obtainedfrom New York State Department of Health). The sera was bound to ProteinA-sepharose in an overnight incubation at 4° C. Following thisincubation period, the material was washed 4× with 1× buffer A. Lysates,precleared with normal human sera and protein A-sepharose, were thenincubated overnight at 4° C. with the HIV1-seropositive human sera boundto protein A-sepharose. After the overnight incubation period, thesamples were washed 4× with 1× buffer A and 2× with a LiCl₂/urea buffer.Precipitated proteins were dissociated from the immune complexes by theaddition of 2× Laemmli's buffer (125 mM Tris (pH6.8), 4% SDS, 20%glycerol, 10% 2-mercaptoethanol) and boiling for 5 min. Proteins werefractionated on a 10% Dreyfuss gel system (Dreyfuss et al., 1984), fixedand treated with 1M Na-salicylate for fluorography. This analysisindicated that HIV1gag and gp120(+transmembrane) gene products wereprecipitated from vCP300-infected cells, but were not precipitated frommock infected or ALVAC-infected cells.

Expression of the nef and pol epitopes in vCP300-infected cells has notbeen confirmed because no epitope specific serological reagents are yetavailable. Nucleotide sequence analysis, however, has confirmed that thenef and pol sequences cloned into vCP300 are correct. PCR fragments,containing the nef and pol expression cassettes, were generated fromvCP300 DNA. Nucleotide sequence analysis of these fragments indicatedthat the nef and pol sequences are correct. vCP205 expresses the samecell surface-associated form of HIV1 gp120 as expressed by vCP300. Theimmunogenicity of this gene product, as expressed by vCP205, has beenassayed in small laboratory animals.

Example 18 Immunogenicity Studies

Groups of two rabbits or guinea pigs were inoculated intramuscularly(im) with 10⁸ pfu of ALVAC, vCP205, or with 0.1 mg of peptide CLTB-36(GPKEPFRDYVDRFYKNKRKRIHIGPGRAFYTTKN) (SEQ ID NOS: 118) adjuvanted with0.05 mg of QS-21 according to the schedule below (Table 21).

TABLE 21 Immunization schedule for rabbits and guinea pigs inoculatedwith ALVAC, vCP205, or with peptide CLTB-36 in QS-21. INOCULATION GROUPWEEK 0 WEEK 4 WEEK 8 1 ALVAC ALVAC CLTB-36/QS-21 2 vCP205 vCP205 vCP2053 vCP205 vCP205 CLTB-36/QS-21 4 CLTB-36/QS-21 CLTB-36/QS-21CLTB-36/QS-21

Each rabbit and guinea pig was bled prior to the first inoculation andat 2-week intervals following the first inoculation through week 14.Serum was prepared from each blood sample and stored at −70° C. untiluse. Each serum was tested for antibody responses to recombinant HIVMN/BRU hybrid gp160 or to 25-mer synthetic HIV MN gp120 V3 loop(American Bio-Technologies, Inc. Cambridge, Mass., product # 686010) bykinetics enzyme-linked immunosorbant assay (KELISA).

Rabbits immunized with vCP205 (Group 2) produced the highest levels ofanti-gp160 antibodies (FIG. 20). Rabbits were immunized according to theschedule in Table 21 (arrows in FIG. 20) and bled at 2 week intervals.Each serum was diluted 1:100 in dilution buffer and tested forreactivity with purified recombinant HIV MN/BRU gp160 using a kineticsELISA. Both rabbits in this group began producing gp160 reactiveantibodies after a single inoculation. Boosting with subsequentinoculations produced only minor increases in antibody levels. Rabbitsinoculated twice with vCP205 and boosted with peptide CLTB-36 in QS-21adjuvant (Group 3) apparently failed to make anti-gp160 antibodies whencompared to control rabbits (Group 1). Of the rabbits immunized threetimes with peptide CLTB-36 in QS-21, only one responded by generatinggp160-specific antibodies. The one responsive rabbit (A353) beganproducing gp160 antibodies only after the third immunization.

Rabbits immunized only with peptide CLTB-36 would not be expected togenerate broadly reactive gp160-specific antibodies since the peptidecontains only a small portion of the envelope glycoprotein, the V3 loop.Thus, sera were tested for reactivity to a peptide containing 25 aminoacids of the HIV MN V3 loop (FIG. 21). Rabbits were immunized accordingto the schedule in Table 21 (arrows in FIG. 22) and bled at 2 weekintervals. Each serum was diluted 1:100 in dilution buffer and testedfor reactivity with a 25-mer synthetic peptide representing the HIV MNgp120 V3 loop (CNKRKRIHIGPGRAFYTTKNIIGTIC; (SEQ ID NO: 119) AmericanBio-Technologies, Inc. Cambridge, Mass., product # 686010) using akinetics ELISA. As before, the highest V3 antibody responses were foundin the sera of rabbits inoculated three times with vCP205. Twoinoculations with vCP205 followed by peptide CLTB-36 in QS-21 producedanti-V3 antibody responses, but not as high as Group 2 rabbits. Also, asbefore, only one rabbit responded to three inoculations with peptideCLTB-36 in QS-21.

Guinea pigs in all groups, including one animal in Group 1, producedantibodies that reacted with HIV gp160 (FIG. 22). Guinea pigs wereimmunized according to the schedule in Table 21 (arrows in FIG. 22) andbled at 2 week intervals. Each serum was diluted 1:100 in dilutionbuffer and tested for reactivity with purified recombinant HIV MN/BRUgp160 using a kinetics ELISA. The single animal in Group 1 thatresponded did so only after inoculation with peptide CLTB-36 in QS-21adjuvant. Antibody levels in the sera of all guinea pigs in Groups 2, 3,and 4 were similar. Most of the guinea pigs responded to a singleinoculation by producing gp160 antibodies.

Similar results were seen using a HIV MN V3 25-mer peptide as the KELISAantigen (FIG. 23). Guinea pigs were immunized according to the schedulein Table 21 (arrows in FIG. 23) and bled at 2 week intervals. Each serumwas diluted 1:100 in dilution buffer and tested for reactivity with a25-mer synthetic peptide representing the HIV MN gp120 V3 loop(CNKRKRIHIGPGRAFYTTKNIIGTIC (SEQ ID NO: 120) American Bio-Technologies,Inc. Cambridge, Mass., product # 686010) using a kinetics ELISA. Asingle inoculation of peptide CLTB-36 in QS-21 elicited V3 antibodyresponses which were boosted by second and third inoculations. Twoinoculations with vCP205 were necessary to induce V3 antibody responseswhich was boosted to higher levels by the third inoculation of vCP205 orCLTB-36 in QS-21.

Expression and immunogenicity of the Nef and Pol CTL epitopes expressedby vCP300 is demonstrated by the following in vitro assays. Fresh PBMCsamples were derived from HIV-seropositive individuals. Twenty-percentof these cells were inoculated with vCP300 at an m.o.i. of 10. Two hourspost-infection the cells were washed and mixed with autologous,uninoculated PBMCs at a ratio of uninoculated/inoculated of 10:1.(Seeding density equaled 1.5×10⁶ cells/ml). On day 0, exogenous IL-7 wasadded at a final concentration of 1000 U/ml. On day three, the additionof an exogenous source of IL-7 and IL-2 was added at a finalconcentration of 1000 U/ml and 220 U/ml, respectively. After 12 days inculture, the in vitro stimulated cell population was used in a standard⁵¹Cr-release assay using autologous Epstein-Barr virus-transformed Bcells infected with vaccinia virus (WR) recombinants expressing HIV-1proteins as targets. The results from assays obtained using anEffector/Target (E/T) cell ratio of 20:1 are shown in FIGS. 24 and 25and expressed as percent specific lysis. The combined resultsdemonstrate the ability of vCP300-infected PBMCs to stimulate HIV-1,Env-, Gag-, Pol-, and Nef-specific cytolytic activity. Further,abrogation of the cytolytic activities by anti-CD8 monoclonal antibodiesdemonstrates that the nature of the cell mediating the cytolyticactivities are classical CD8⁺ CTLs.

In summary, the inoculation of rabbits and guinea pigs with the HIVALVAC recombinant canarypox virus, vCP205, elicited antibodies to theHIV envelope glycoprotein and a region of the HIV envelope glycoproteinassociated with neutralization of HIV, the gp120 V3 loop region. Theexpression and immunogenicity of the vCP300 expressed Env, Gag, Pol andNef encoded products is demonstrated by the in vitro stimulation of CD8⁺CTLs from seropositive individuals. Thus, vCP205 and vCP300 andprecursors to these recombinants and expression products and DNA fromthese recombinants are useful, as described above.

Example 19 Generation of vCP1307; an ALVAC Recombinant Expressing a Formof HIV1 gp120+TM with 2 ELDKWA Epitopes Inserted into the gp120 V3 Loop

vCP1307, an ALVAC recombinant expressing HIV1 gp120+TM with 2 ELDKWAepitopes from HIV1 gp41 inserted into the gp120 V3 loop region, wasgenerated by the following procedure. The sequence encoding part of theELDKWA elements and V3 loop was cloned into pBSK+ (Stratagene, LaJolla,Calif.). This was accomplished by cloning a 225 bp EcoRI-SacI-digestedPCR fragment, containing part of the ELDKWA-V3 loop sequence, into the2,900 bp EcoRI-SacI fragment of pBSK+. (This PCR fragment was generatedfrom the plasmid, pBSHIVMN120T, with the primers, HIVP72 (SEQ ID NO:121; 5′-TTATTACCATTCCAAGTACTATT-3′) and HIVP74 (SEQ ID NO: 122)5′-TCTGTACAAATTAATTGTACAAGACCCAACTACGAGCTCGACAAATGGGCCCATATAGGACCAGGGAGAGAATTGGATAAGTGGGCGAATATAATAGGAACTATAAGAC-3′).) The plasmidgenerated by this manipulation is called pHIV55.

pBSHIVMN120T, a plasmid containing the H6-promoted HIV1 gp120+TM gene,was generated by the following procedure. A plasmid, pMN1.8-9,containing a cDNA copy of the HIV1 (MN) env gene, was obtained fromMarvin Reitz (NCI, NIH). An early transcription termination signalsequence, T₅NT, in the env gene was modified. This was accomplished bycloning a 1,100 bp KpnI-EcoRI-digested PCR fragment, containing theT₅NT-modified 5′-end of the env gene, into the 2,900 bp KpnI-EcoRIfragment of pBSK+. (This PCR fragment was generated from the plasmid,pMN1.8-9, with the oligonucleotides, HIVMN6 (SEQ ID NO: 123;5′-GGGTTATTAATGATCTGTAG-3′) and HIV3B2 (SEQ ID NO: 124;5′-GAATTACAGTAGAAGAATTCCCCTCCACAATTAAAAC-3′).) The plasmid generated bythis manipulation is called pBSMIDMN.

The T₅NT-modified 5′-end of the env gene was then cloned upstream to therest of the env gene. This was accomplished by cloning the 1,025 bpKpnI-EcoRI fragment of pBSMIDMN, containing the T₅NT-modified 5′-end ofthe env gene, into the 4,300 bp KpnI-EcoRI fragment of pBS3MN. (pBS3MNwas generated by cloning a 430 bp EcoRI-SacI-digested PCR fragment,containing a central portion of the env gene, and a 1,050 bpSacI-XbaI-digested PCR fragment, containing the 3′-end of the env gene,into the 2,900 bp EcoRI-XbaI fragment of pBSK+. The 430 bp PCR fragmentwas generated from the plasmid, pMN1.8-9, with the oligonucleotides,HIV3B1 (SEQ ID NO: 125; 5′-GTTTTAATTGTGGAGGGGAATTCTTCTACTGTAATTC-3′) andHIVMN4 (SEQ ID NO: 126; 5′-ATCATCGAGCTCCTATCGCTGCTC-3′). The 1,050 bpPCR fragment was generated from the plasmid, pMN1.8-9, with theoligonucleotides, HIVMN5 (SEQ ID NO: 127;5′-ATCATCGAGCTCTGTTCCTTGGGTTCTTAG-3′) and HIVMN3P (SEQ ID NO: 128;5′-ATCATCTCTAGAATAAAAATTATAGCAAAGCCCTTTCCAAGCC-3′).) The plasmidgenerated by this manipulation is called pBSMID3MN.

The H6 promoter (Perkus et al., 1989) was then cloned upstream to theenv gene. This was accomplished by cloning the 320 bp KpnI fragment ofpH6IIIBE, containing the H6 promoter linked to the 5′-end of the HIV1(IIIB) env gene, and the 2,450 bp KpnI-XbaI fragment of pBSMID3MN,containing the bulk of the HIV1 (MN) env gene, into the 2,900 bpKpnI-XbaI fragment of pBSK+. The plasmid generated by this manipulationis called pH6HMNE.

The sequence encoding gp41 was then replaced with the sequence encodingthe HIV1 env transmembrane (TM) region. This was accomplished by cloninga 480 bp EcoRI-XbaI-digested PCR fragment, containing the 3′-end of thegp120 gene and the HIV1 env transmembrane region, into the 4,200 bpEcoRI-XbaI fragment of pH6HMNE. (This PCR fragment was generated fromthe PCR fragment, PCR-MN11, and oligonucleotides, HIVTM1 (SEQ ID NO:129; 5′-TTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTCTCTGTAGTGAATAGAGTTAGGCAGGGATAA-3′) and HIVTM2 (SEQ ID NO: 130;5′-TTATCCCTGCCTAACTCTATTCACTACAGAGAGTACAGCAAAAACTATTCTTAAACCTACCAAGCCTCCTACTATCATTATGAATAA-3′), with the oligonucleotides, HIV3B1 (SEQ IDNO: 125) and HIVTM3 (SEQ ID NO: 131;5′-ATCATCTCTAGAATAAAAATTATCCCTGCCTAACTCTATTCAC-3′). PCR-MN11 wasgenerated from the plasmid, pH6HMNE, with the oligonucleotides, HIV3B1(SEQ ID NO: 125) and HIVMN18 (SEQ ID NO: 132;5′-GCCTCCTACTATCATTATGAATAATCTTTTTTCTCTCTG-3′).) The plasmid generatedby this manipulation is called pBSHIVMN120T.]

Another part of the sequence encoding the ELDKWA epitopes and V3 loopwas then cloned into PBSK+. This was accomplished by cloning a 300 bpHindIII-SacI-digested PCR fragment, containing part of the ELDKWA-V3loop sequence, into the 2,900 bp HindIII-SacI fragment of PBSK+. (ThisPCR fragment was generated from the plasmid, pBSHIVMN120T, with theprimers, HIVP69 (SEQ ID NO: 133; 5′-TGATAGTACCAGCTATAGGTTGAT-3′) andHIVP75 (SEQ ID NO: 134; 5′-TTTGTCGAGCTCGTAGTTGGGTCTTGTACAATT-3′).) Theplasmid generated by this manipulation is called pHIV56.

The ELDKWA-V3 loop sequences from pHIV55 and pHIV56 were then clonedinto the H6-promoted gp120+TM gene. This was accomplished by cloning the225 bp EcoRI-SacI fragment of pHIV55 and the 300 bp HindIII-SacIfragment of pHIV56, containing the ELDKWA-V3 loop sequences, into the4,300 bp EcoRI-HindIII fragment of pBSHIVMN120T. The plasmid generatedby this manipulation is called pHIV57.

The H6-promoted gp120+TM construct containing the ELDKWA epitopes wasthen cloned between C5 flanking arms. This was accomplished by cloningthe 1,700 bp NruI-XbaI fragment of pHIV57, containing the H6-promotedgp120+TM (with ELDKWA epitopes) gene, into the 4,700 bp NruI-XbaIfragment of pSIVGC15. (pSIVGC15 contains the H6-promoted SIV env genecloned between C5 flanking arms.) The plasmid generated by thismanipulation is called pHIV59. The DNA sequence of the H6-promotedgp120+TM (with ELDKWA epitopes) gene in pHIV59 is shown in FIG. 26.pHIV59 was used in in vitro recombination experiments with ALVAC as therescuing virus to yield vCP1307.

FACS analysis was performed to determine whether HIV1 gp120+TM (withELDKWA epitopes) was expressed on the surface of vCP1307-infected cells.5×10⁶ HeLa-S3 cells in S-MEM (Sigma M-8028 Joklik suspension media) wereinfected at an m.o.i. of 5 pfu/cell with ALVAC, vP1286 (a WR recombinantexpressing HIV1 gp120+TM) or vCP1307. Following a 60 minute adsorptionperiod at 37° C., the cells were washed with 10 mls of S-MEM andcentrifuged at 1,000 RPM for 5 minutes. The samples were thenresuspended in 1 ml of S-MEM, transferred to 5 ml Sarstadt tubes andplaced on a rotator at 37° C. After 18 hours, 200 ul aliquots (1×10⁶cells) were placed in polypropylene tubes and washed with 3 mls ofPBS-CMF (with 0.2% NaN₃+0.2% BSA). The supernatant was then decanted andthe pellet was resuspended in 100 ul of a 1:100 dilution of sera fromHIV1-seropositive humans (obtained from the New York State Dept. ofHealth) or a 1:100 dilution of a human monoclonal antibody specific forthe ELDKWA epitope, IAM41-2F5 (obtained from Viral Testing SystemsCorp., Houston, Tex.). The samples were incubated at 4° C. for 60minutes, washed two times with PBS-CMF (with 0.2% NaN₃+0.2% BSA) andcentrifuged at 1,000 RPM for 5 minutes. The supernatant was decanted andthe pellet was resuspended in a 1:100 dilution of goat anti-human FITC(obtained from Boehringer Mannheim). The samples were incubated at 4° C.for 30 minutes, washed twice with PBS-CMF (with 0.2% NaN₃+0.2% BSA) andanalyzed on a Facscan flow cytometer. A gene product containing theELDKWA epitope was expressed at low levels on the surface ofvCP1307-infected cells, but was not expressed on the surface ofALVAC-infected or vP1286-infected cells (FIG. 27, lower panel). A geneproduct reactive with the HIV1-seropositive sera was expressed on thesurface of vP1286-infected cells and vCP1307-infected cells, but was notexpressed on the surface of ALVAC-infected cells (FIG. 27, upper panel).These results indicate that the ELDKWA epitope of the HIV1 gp120+TM(with ELDKWA epitopes) gene product is expressed on the surface ofvCP1307-infected cells, consistent with the fact that a portion of theV3 loop of this gene product has been replaced with ELDKWA epitopes.

Immunoprecipitation analysis was performed to determine whether vCP1307expresses a form of gp120+TM which contains an immunogenic ELDKWAepitope. HeLa cell monolayers were infected at an m.o.i. of 10 pfu/cellwith ALVAC (the parental virus), vP1286 (a WR recombinant expressingHIV1 gp120+TM) or vCP1307. Following an hour adsorption period, theinoculum was removed and the cells were overlayed with 2 mls of modifiedEagle's medium (minus cysteine and methionine) containing 2% dialyzedfetal bovine serum and [³⁵S]-TRANS label (30 μCi/ml). The lysates wereharvested at 18 hrs post-infection by addition of 1 ml 3× buffer A (450mM NaCl, 3% NP-40, 30 mM Tris (pH=7.4), 3 mM EDTA, 0.03% Na-Azide and0.6 mg/ml PMSF) and analyzed for expression of 1) the ELDKWA epitope,using a 1:100 dilution of a human monoclonal antibody specific for theELDKWA epitope, IAM41-2F5 (obtained from Viral Testing Systems Corp.,Houston, Tex.) and 2) HIV1 gene products, using a 1:100 dilution of serafrom HIV1-seropositive humans (obtained from the New York State Dept. ofHealth). Lysates, precleared with normal human sera and a proteinA-sepharose complex, were incubated overnight at 4° C. with anIAM41-2F5-protein A-sepharose complex or an HIV1-seropositivesera-protein A-sepharose complex. The samples were washed 4× with 1×buffer A and 2× with a LiCl₂/urea buffer. Precipitated proteins weredissociated from the immune complexes by the addition of 2× Laemmli'sbuffer (125 mM Tris (pH=6.8), 4% SDS, 20% glycerol, 10%2-mercaptoethanol) and boiling for 5 min. Proteins were fractionated onan SDS-polyacrylamide gel, fixed and treated with 1 M Na-salicylate forfluorography. Proteins of the appropriate size were precipitated withthe monoclonal antibody to the ELDKWA epitope from vCP1307-infectedcells, but were not precipitated from ALVAC-infected cells orvP1286-infected cells. Furthermore, proteins of the appropriate sizewere precipitated with the human HIV1-seropositive sera fromvP1286-infected cells and vCP1307-infected cells, but were notprecipitated from ALVAC-infected cells. These results indicate thatvCP1307 expresses a form of gp120+TM which contains an antigenic ELDKWAepitope.

Example 20 Generation of vP1313; a NYVAC Recombinant Expressing a Formof HIV1 gp120+TM with 2 ELDKWA Epitopes Inserted into the gp120 V3 Loop

vP1313, a NYVAC recombinant expressing HIV1 gp120+TM with 2 ELDKWAepitopes from HIV1 gp41 inserted into the gp120 V3 loop, was generatedby the following procedure. The sequence encoding part of the ELDKWAepitopes and V3 loop was cloned into pBSK+. This was accomplished bycloning a 225 bp EcoRI-SacI-digested PCR fragment, containing part ofthe ELDKWA-V3 loop sequence, into the 2,900 bp EcoRI-SacI fragment ofpBSK+. (This PCR fragment was generated from the plasmid, pBSHIVMN120T,with the primers, HIVP72 (SEQ ID NO: 144; 5′-TTATTACCATTCCAAGTACTATT-3′)and HIVP74 (SEQ ID NO: 145;5′-TCTGTACAAATTAATTGTACAAGACCCAACTACGAGCTCGACAAATGGGCCCATATAGGACCAGGGAGAGAATTGGATAAGTGGGCGAATATAATAGGAACTATAAGAC-3′).) The plasmidgenerated by this manipulation is called pHIV55.

pBSHIVMN120T, a plasmid containing the H6-promoted HIV1 gp120+TM gene,was generated by the following procedure. A plasmid, pMN1.8-9,containing a cDNA copy of the HIV1 (MN) env gene, was obtained fromMarvin Reitz (NCI, NIH). An early transcription termination signalsequence, T₅NT, in the env gene was modified. This was accomplished bycloning a 1,100 bp KpnI-EcoRI-digested PCR fragment, containing theT₅NT-modified 5′-end of the env gene, into the 2,900 bp KpnI-EcoRIfragment of pBSK+. (This PCR fragment was generated from the plasmid,pMN1.8-9, with the oligonucleotides, HIVMN6 (SEQ ID NO: 146;5′-GGGTTATTAATGATCTGTAG-3′) and HIV3B2 (SEQ ID NO: 124;5′-GAATTACAGTAGAAGAATTCCCCTCCACAATTAAAAC-3′).) The plasmid generated bythis manipulation is called pBSMIDMN.

The T₅NT-modified 5%-end of the env gene was then cloned upstream to therest of the env gene. This was accomplished by cloning the 1,025 bpKpnI-EcoRI fragment of pBSMIDMN, containing the T₅NT-modified 5′-end ofthe env gene, into the 4,300 bp KpnI-EcoRI fragment of pBS3MN. (pBS3MNwas generated by cloning a 430 bp EcoRI-SacI-digested PCR fragment,containing a central portion of the env gene, and a 1,050 bpSacI-XbaI-digested PCR fragment, containing the 3′-end of the env gene,into the 2,900 bp EcoRI-XbaI fragment of pBSK+. The 430 bp PCR fragmentwas generated from the plasmid, pMN1.8-9, with the oligonucleotides,HIV3B1 (SEQ ID NO: 125; 5′-GTTTTAATTGTGGAGGGGAATTCTTCTACTGTAATTC-3′) andHIVMN4 (SEQ ID NO: 126; 5′-ATCATCGAGCTCCTATCGCTGCTC-3′). The 1,050 bpPCR fragment was generated from the plasmid, pMN1.8-9, with theoligonucleotides, HIVMN5 (SEQ ID NO: 127;5′-ATCATCGAGCTCTGTTCCTTGGGTTCTTAG-3′) and HIVMN3P (SEQ ID NO: 128;5′-ATCATCTCTAGAATAAAAATTATAGCAAAGCCCTTTCCAAGCC-3′).) The plasmidgenerated by this manipulation is called pBSMID3MN.

The H6 promoter was then cloned upstream to the env gene. This wasaccomplished by cloning the 320 bp KpnI fragment of pH6IIIBE, containingthe H6 promoter linked to the 5′-end of the HIV1 (IIIB) env gene, andthe 2,450 bp KpnI-XbaI fragment of pBSMID3MN, containing the bulk of theHIV1 (MN) env gene, into the 2,900 bp KpnI-XbaI fragment of pBSK+. Theplasmid generated by this manipulation is called pH6HMNE.

The sequence encoding gp41 was then replaced with the sequence encodingthe HIV1 env transmembrane (TM) region. This was accomplished by cloninga 480 bp EcoRI-XbaI-digested PCR fragment, containing the 3′-end of thegp120 gene and the HIV1 env transmembrane region, into the 4,200 bpEcoRI-XbaI fragment of pH6HMNE. (This PCR fragment was generated fromthe PCR fragment, PCR-MN11, and oligonucleotides, HIVTM1 (SEQ ID NO:129; 5′-TTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTCTCTGTAGTGAATAGAGTTAGGCAGGGATAA-3′) and HIVTM2 (SEQ ID NO: 130;5′-TTATCCCTGCCTAACTCTATTCACTACAGAGAGTACAGCAAAAACTATTCTTAAACCTACCAAGCCTCCTACTATCATTATGAATAA-3′), with the oligonucleotides, HIV3B1 (SEQ IDNO: 125) and HIVTM3 (SEQ ID NO: 131;5′-ATCATCTCTAGAATAAAAATTATCCCTGCCTAACTCTATTCAC-3′). PCR-MN11 wasgenerated from the plasmid, pH6HMNE, with the oligonucleotides, HIV3B1(SEQ ID NO: 125) and HIVMN18 (SEQ ID NO: 132;5′-GCCTCCTACTATCATTATGAATAATCTTTTTTCTCTCTG-3′).) The plasmid generatedby this manipulation is called pBSHIVMN120T.]

Another part of the sequence encoding the ELDKWA epitopes and V3 loopwas then cloned into pBSK+. This was accomplished by cloning a 300 bpHindIII-SacI-digested PCR fragment, containing part of the ELDKWA-V3loop sequence, into the 2,900 bp HindIII-SacI fragment of pBSK+. (ThisPCR fragment was generated from the plasmid, pBSHIVMN120T, with theprimers, HIVP69 (SEQ ID NO: 133; 5′-TGATAGTACCAGCTATAGGTTGAT-3′) andHIVP75 (SEQ ID NO: 134; 5′-TTTGTCGAGCTCGTAGTTGGGTCTTGTACAATT-3′).) Theplasmid generated by this manipulation is called pHIV56.

The ELDKWA-V3 loop sequences from pHIV55 and pHIV56 were then clonedinto the H6-promoted gp120+TM gene. This was accomplished by cloning the225 bp EcoRI-SacI fragment of pHIV55 and the 300 bp HindIII-SacIfragment of pHIV56, containing the ELDKWA-V3 loop sequences, into the4,300 bp EcoRI-HindIII fragment of pBSHIVMN120T. The plasmid generatedby this manipulation is called pHIV57.

The H6-promoted gp120+TM construct containing the ELDKWA epitopes wasthen cloned between C5 flanking arms. This was accomplished by cloningthe 1,700 bp NruI-XbaI fragment of pHIV57, containing the H6-promotedgp120+TM (with ELDKWA epitopes) gene, into the 4,700 bp NruI-XbaIfragment of pSIVGC15. (pSIVGC15 contains the H6-promoted SIV env genecloned between C5 flanking arms) The plasmid generated by thismanipulation is called pHIV59.

The H6-promoted gp120+TM (with ELDKWA epitopes) gene was then clonedbetween I4L flanking arms. This was accomplished by cloning the 1,850 bpBamHI-SmaI fragment of pHIV59, containing the H6-promoted gp120+TM (withELDKWA epitopes) gene, into the 3,600 bp BamHI-SmaI fragment ofpSD550VC. The plasmid generated by this manipulation is called pHIV60.The DNA sequence of the H6-promoted gp120+TM (with ELDKWA epitopes) genein pHIV60 is shown in FIG. 28.

pHIV60 was used in in vitro recombination experiments with NYVAC as therescuing virus to yield vP1313.

FACS analysis was performed to determine whether HIV1 gp120+TM (withELDKWA epitopes) was expressed on the surface of vP1313-infected cells.5×10⁶ HeLa-S3 cells in S-MEM (Sigma M-8028 Joklik suspension media) wereinfected at an m.o.i. of 5 pfu/cell with NYVAC, vP1286 (a WR recombinantexpressing HIV1 gp120+TM) or vP1313. Following a 60 minute adsorptionperiod at 37° C., the cells were washed with 10 mls of S-MEM andcentrifuged at 1,000 RPM for 5 minutes. The samples were thenresuspended in 1 ml of S-MEM, transferred to 5 ml Sarstadt tubes andplaced on a rotator at 37° C. After 18 hours, 200 ul aliquots (1×10⁶cells) were placed in polypropylene tubes and washed with 3 mls ofPBS-CMF (with 0.2% NaN₃+0.2% BSA). The supernatant was then decanted andthe pellet was resuspended in 100 ul of a 1:100 dilution of sera fromHIV1-seropositive humans (obtained from the New York State Dept. ofHealth), or a 1:100 dilution of a human monoclonal antibody specific forthe ELDKWA epitope, IAM41-2F5 (obtained from Viral Testing Systems Corp.Houston, Tex.). The samples were incubated at 4° C. for 60 minutes,washed two times with PBS-CMF (with 0.2% NaN₃+0.2% BSA) and centrifugedat 1,000 RPM for 5 minutes. The supernatant was decanted and the pelletwas resuspended in a 1:100 dilution of goat anti-human FITC (obtainedfrom Boehringer Mannheim). The samples were incubated at 4° C. for 30minutes, washed twice with PBS-CMF (with 0.2% NaN₃+0.2% BSA) andanalyzed on a Facscan flow cytometer. A gene product containing theELDKWA epitope was expressed on the surface of vP1313-infected cells,but was not expressed on the surface of NYVAC-infected orvP1286-infected cells (FIG. 29, lower panel). A gene product reactivewith the HIV1-seropositive sera was expressed on the surface ofvP1286-infected cells and vP1313-infected cells, but was not expressedon the surface of NYVAC-infected cells (FIG. 29, upper panel). Theseresults indicate that the ELDKWA epitope of the HIV1 gp120+TM (withELDKWA epitopes) gene product is expressed on the surface ofvP1313-infected cells, consistent with the fact that a portion of the V3loop of this gene product has been replaced with ELDKWA epitopes.

Immunoprecipitation analysis was performed to determine: whether vP1313expresses a form of gp120+TM which contains an immunogenic ELDKWAepitope. HeLa cell monolayers were infected at an m.o.i. of 10 pfu/cellwith NYVAC (the parental virus), vP1286 (a WR recombinant expressingHIV1 gp120+TM) or vP1313. Following an hour adsorption period, theinoculum was removed and the cells were overlayed with 2 mls of modifiedEagle's medium (minus cysteine and methionine) containing 2% dialyzedfetal bovine serum and [³⁵S]-TRANS label (30 μCi/ml). The lysates wereharvested at 18 hrs post-infection by addition of 1 ml 3× buffer A (450mM NaCl, 3% NP-40, 30 mM Tris (pH=7.4), 3 mM EDTA, 0.03% Na-Azide and0.6 mg/ml PMSF) and analyzed for expression of 1) the ELDKWA epitope,using a 1:100 dilution of a human monoclonal antibody specific for theELDKWA epitope, IAM41-2F5 (obtained from Viral Testing Systems Corp.,Houston, Tex.) and 2) HIV1 gene products, using a 1:100 dilution of serafrom HIV1-seropositive humans (obtained from the New York State Dept. ofHealth). Lysates, precleared with normal human sera and a proteinA-sepharose complex, were incubated overnight at 4° C. with anIAM41-2F5-protein A-sepharose complex or an HIV1-seropositivesera-protein A-sepharose complex. The samples were washed 4× with 1×buffer A and 2× with a LiCl₂/urea buffer. Precipitated proteins weredissociated from the immune complexes by the addition of 2× Laemmli'sbuffer (125 mM Tris (pH=6.8), 4% SDS, 20% glycerol, 10%2-mercaptoethanol) and boiling for 5 min. Proteins were fractionated onan SDS-polyacrylamide gel, fixed and treated with 1 M Na-salicylate forfluorography. Proteins of the appropriate size were precipitated withthe monoclonal antibody to the ELDKWA epitope from vP1313-infectedcells, but were not precipitated from NYVAC-infected cells orvP1286-infected cells. Furthermore, proteins of the appropriate sizewere precipitated with the human HIV1-seropositive sera fromvP1286-infected cells and vP1313-infected cells, but were notprecipitated from NYVAC-infected cells. These results indicate thatvP1313 expresses a form of gp120+TM which contains an immunogenic ELDKWAepitope.

Example 21 Generation of vP1319; a COPAK Recombinant Expressing a Formof HIV1 gp120+TM with 2 ELDKWA Epitopes Inserted into the gp120 V3 Loop

vP1319, a COPAK recombinant expressing HIV1 gp120+TM with 2 ELDKWAepitopes from HIV1 gp41 inserted into the gp120 V3 loop, was generatedby the following procedure. The sequence encoding part of the ELDKWAepitopes and V3 loop was cloned into pBSK+. This was accomplished bycloning a 225 bp EcoRI-SacI-digested PCR fragment, containing part ofthe ELDKWA-V3 loop sequence, into the 2,900 bp EcoRI-SacI fragment ofpBSK+. (This PCR fragment was generated from the plasmid, pBSHIVMN120T,with the primers, HIVP72 (SEQ ID NO: 121; 5′-TTATTACCATTCCAAGTACTATT-3′)and HIVP74 (SEQ ID NO: 122;5′-TCTGTACAAATTAATTGTACAAGACCCAACTACGAGCTCGACAAATGGGCCCATATAGGACCAGGGAGAGAATTGGATAAGTGGGCGAATATAATAGGAACTATAAGAC-3′).) The plasmidgenerated by this manipulation is called pHIV55. pBSHIVMN120T, a plasmidcontaining the H6-promoted HIV1 gp120+TM gene, was generated by thefollowing procedure. A plasmid, pMN1.8-9, containing a cDNA copy of theHIV1 (MN) env gene, was obtained from Marvin Reitz (NCI, NIH). An earlytranscription termination signal sequence, T₅NT, in the env gene wasmodified. This was accomplished by cloning a 1,100 bpKpnI-EcoRI-digested PCR fragment, containing the T₅NT-modified 5′-end ofthe env gene, into the 2,900 bp KpnI-EcoRI fragment of pBSK+. (This PCRfragment was generated from the plasmid, pMN1.8-9, with theoligonucleotides, HIVMN6 (SEQ ID NO: 123; 5′-GGGTTATTAATGATCTGTAG-3′)and HIV3B2 (SEQ ID NO: 124;5′-GAATTACAGTAGAAGAATTCCCCTCCACAATTAAAAC-3′).) The plasmid generated bythis manipulation is called pBSMIDMN.

The T₅NT-modified 5′-end of the env gene was then cloned upstream to therest of the env gene. This was accomplished by cloning the 1,025 bpKpnI-EcoRI fragment of pBSMIDMN, containing the T₅NT-modified 5′-end ofthe env gene, into the 4,300 bp KpnI-EcoRI fragment of pBS3MN. (pBS3MNwas generated by cloning a 430 bp EcoRI-SacI-digested PCR fragment,containing a central portion of the env gene, and a 1,050 bpSacI-XbaI-digested PCR fragment, containing the 3′-end of the env gene,into the 2,900 bp EcoRI-XbaI fragment of pBSK+. The 430 bp PCR fragmentwas generated from the plasmid, pMN1.8-9, with the oligonucleotides,HIV3B1 (SEQ ID NO: 125; 5′-GTTTTAATTGTGGAGGGGAATTCTTCTACTGTAATTC-3′) andHIVMN4 (SEQ ID NO: 126; 5′-ATCATCGAGCTCCTATCGCTGCTC-3′). The 1,050 bpPCR fragment was generated from the plasmid, pMN1.8-9, with theoligonucleotides, HIVMN5 (SEQ ID NO: 127;5′-ATCATCGAGCTCTGTTCCTTGGGTTCTTAG-3′) and HIVMN3P (SEQ ID NO: 128;5′-ATCATCTCTAGAATAAAAATTATAGCAAAGCCCTTTCCAAGCC-3′).) The plasmidgenerated by this manipulation is called pBSMID3MN.

The H6 promoter was then cloned upstream to the env gene. This wasaccomplished by cloning the 320 bp KpnI fragment of pH6IIIBE, containingthe H6 promoter linked to the 5′-end of the HIV1 (IIIB) env gene, andthe 2,450 bp KpnI-XbaI fragment of pBSMID3MN, containing the bulk of theHIV1 (MN) env gene, into the 2,900 bp KpnI-XbaI fragment of pBSK+. Theplasmid generated by this manipulation is called pH6HMNE.

The sequence encoding gp41 was then replaced with the sequence encodingthe HIV1 env transmembrane (TM) region. This was accomplished by cloninga 480 bp EcoRI-XbaI-digested PCR fragment, containing the 3′-end of thegp120 gene and the HIV1 env transmembrane region, into the 4,200 bpEcoRI-XbaI fragment of pH6HMNE. (This PCR fragment was generated fromthe PCR fragment, PCR-MN11, and oligonucleotides, HIVTM1 (SEQ ID NO:129; 5′-TTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTCTCTGTAGTGAATAGAGTTAGGCAGGGATAA-3′) and HIVTM2 (SEQ ID NO: 130;5′-TTATCCCTGCCTAACTCTATTCACTACAGAGAGTACAGCAAAAACTATTCTTAAACCTACCAAGCCTCCTACTATCATTATGAATAA-3′), with the oligonucleotides, HIV3B1 (SEQ IDNO: 125) and HIVTM3 (SEQ ID NO: 131;5′-ATCATCTCTAGAATAAAAATTATCCCTGCCTAACTCTATTCAC-3′). PCR-MN11 wasgenerated from the plasmid, pH6HMNE, with the oligonucleotides, HIV3B1(SEQ ID NO: 125) and HIVMN18 (SEQ ID NO: 132;5′-GCCTCCTACTATCATTATGAATAATCTTTTTTCTCTCTG-3′).) The plasmid generatedby this manipulation is called pBSHIVMN120T.

Another part of the sequence encoding the ELDKWA epitopes and V3 loopwas then cloned into pBSK+. This was accomplished by cloning a 300 bpHindIII-SacI-digested PCR fragment, containing part of the ELDKWA-V3loop sequence, into the 2,900 bp HindIII-SacI fragment of pBSK+. (ThisPCR fragment was generated from the plasmid, pBSHIVMN120T, with theprimers, HIVP69 (SEQ ID NO: 133; 5′-TGATAGTACCAGCTATAGGTTGAT-3′) andHIVP75 (SEQ ID NO: 134; 5′-TTTGTCGAGCTCGTAGTTGGGTCTTGTACAATT-3′).) Theplasmid generated by this manipulation is called pHIV56.

The ELDKWA-V3 loop sequences from pHIV55 and pHIV56 were then clonedinto the H6-promoted gp120+TM gene. This was accomplished by cloning the225 bp EcoRI-SacI fragment of pHIV55 and the 300 bp HindIII-SacIfragment of pHIV56, containing the ELDKWA-V3 loop sequences, into the4,300 bp EcoRI-HindIII fragment of pBSHIVMN120T. The plasmid generatedby this manipulation is called pHIV57.

The H6-promoted gp120+TM construct containing the ELDKWA epitopes wasthen cloned between C5 flanking arms. This was accomplished by cloningthe 1,700 bp NruI-XbaI fragment of pHIV57, containing the H6-promotedgp120+TM (with ELDKWA epitopes) gene, into the 4,700 bp NruI-XbaIfragment of pSIVGC15. (pSIVGC15 contains the H6-promoted SIV env genecloned between C5 flanking arms.) The plasmid generated by thismanipulation is called pHIV59.

The H6-promoted gp120+TM (with ELDKWA epitopes) gene was then clonedbetween COPAK flanking arms. This was accomplished by cloning the 1,850bp BamHI-SmaI fragment of pHIV59, containing the H6-promoted gp120+TM(with ELDKWA epitopes) gene, into the 4,600 bp BamHI-SmaI fragment ofpSD553VC. The plasmid generated by this manipulation is called pHIV61.The DNA sequence of the H6-promoted gp120+TM (with ELDKWA epitopes) genein pHIV61 is shown in FIG. 30.

pHIV61 was used in in vitro recombination experiments with COPAK as therescuing virus to yield vP1319.

FACS analysis was performed to determine whether HIV1 gp120+TM (withELDKWA epitopes) was expressed on the surface of vP1319-infected cells.5×10⁶ HeLa-S3 cells in S-MEM (Sigma M-8028 Joklik suspension media) wereinfected at an m.o.i. of 5 pfu/cell with WR, vP1286 (a WR recombinantexpressing HIV1 gp120+TM) or vP1319. Following a 60 minute adsorptionperiod at 37° C., the cells were washed with 10 mls of S-MEM andcentrifuged at 1,000 RPM for 5 minutes. The samples were thenresuspended in 1 ml of S-MEM, transferred to 5 ml Sarstadt tubes andplaced on a rotator at 37° C. After 18 hours, 200 ul aliquots (1×10⁶cells) were placed in polypropylene tubes and washed with 3 mls ofPBS-CMF (with 0.2% NaN₃+0.2% BSA). The supernatant was then decanted andthe pellet was resuspended in 100 ul of a 1:100 dilution of sera fromHIV1-seropositive humans (obtained from the New York State Dept. ofHealth), a 1:500 dilution of a mouse monoclonal antibody specific forthe HIV1 (MN) V3 loop, 50.1 (obtained from M. Robert-Guroff, NCI, NIH.)or a 1:100 dilution of a human monoclonal antibody specific for theELDKWA epitope, IAM41-2F5 (obtained from Viral Testing Systems Corp.,Houston, Tex.). The samples were incubated at 4° C. for 60 minutes,washed two times with PBS-CMF (with 0.2% NaN₃+0.2% BSA) and centrifugedat 1,000 RPM for 5 minutes. The supernatant was decanted and the pelletwas resuspended in a 1:100 dilution of goat anti-human FITC or goatanti-mouse FITC (obtained from Boehringer Mannheim). The samples wereincubated at 4° C. for 30 minutes, washed twice with PBS-CMF (with 0.2%NaN₃+0.2% BSA) and analyzed on a Facscan flow cytometer. A gene productcontaining the ELDKWA epitope was expressed on the surface ofvP1319-infected cells, but was not expressed on the surface ofWR-infected cells or vP1286-infected cells (FIG. 31, middle panel). Agene product containing the V3 loop was expressed on the surface ofvP1286-infected cells, but was not expressed on the surface ofWR-infected cells or vP1319-infected cells (FIG. 31, lower panel). Agene product reactive with the HIV1-seropositive sera was expressed onthe surface of vP1286-infected cells and vP1319-infected cells, but wasnot expressed on the surface of WR-infected cells (FIG. 31, upperpanel). This analysis indicated that 1) the ELDKWA epitope of the HIV1gp120+TM (with ELDKWA epitopes) gene product is expressed on the surfaceof vP1319-infected cells and 2) the gene product expressed on thesurface of vP1319-infected cells does not contain a wild-type V3 loop,consistent with the fact that the V3 loop of this gene product has beenreplaced with ELDKWA epitopes.

Immunoprecipitation analysis was performed to determine whether vP1319expresses a form of gp120+TM which contains an immunogenic ELDKWAepitope. HeLa cell monolayers were infected at an m.o.i. of 10 pfu/cellwith NYVAC (the parental virus), vP1286 (a WR recombinant expressingHIV1 gp120+TM) or vP1319. Following an hour adsorption period, theinoculum was removed and the cells were overlayed with 2 mls of modifiedEagle's medium (minus cysteine and methionine) containing 2% dialyzedfetal bovine serum and [³⁵S]-TRANS label (30 μCi/ml). The lysates wereharvested at 18 hrs post-infection by addition of 1 ml 3× buffer A (450mM NaCl, 3% NP-40, 30 mM Tris (pH=7.4), 3 mM EDTA, 0.03% Na-Azide and0.6 mg/ml PMSF) and analyzed for expression of 1) the ELDKWA epitope,using a 1:100 dilution of a human monoclonal antibody specific for theELDKWA epitope, IAM41-2F5 (obtained from Viral Testing Systems Corp.,Houston, Tex.) and 2) HIV1 gene products, using a 1:100 dilution of serafrom HIV1-seropositive humans (obtained from the New York State Dept. ofHealth). Lysates, precleared with normal human sera and a proteinA-sepharose complex, were incubated overnight at 4° C. with anIAM41-2F5-protein A-sepharose complex or an HIV1-seropositivesera-protein A-sepharose complex. The samples were washed 4× with 1×buffer A and 2× with a LiCl₂/urea buffer. Precipitated proteins weredissociated from the immune complexes by the addition of 2× Laemmli'sbuffer (125 mM Tris (pH=6.8), 4% SDS, 20% glycerol, 10%2-mercaptoethanol) and boiling for 5 min. Proteins were fractionated onan SDS-polyacrylamide gel, fixed and treated with 1 M Na-salicylate forfluorography. Proteins of the appropriate size were precipitated withthe monoclonal antibody to the ELDKWA epitope from vP1319-infectedcells, but were not precipitated from NYVAC-infected cells orvP1286-infected cells. Furthermore, proteins of the appropriate sizewere precipitated with the human HIV1-seropositive sera fromvP1286-infected cells and vP1319-infected cells, but were notprecipitated from NYVAC-infected cells. These results indicate thatvP1319 expresses a form of gp120+TM which contains an immunogenic ELDKWAepitope.

Since vCP1307, vP1313 and vP1319 each express the ELDKWA epitope in animmunogenic configuration, these recombinants have numerous utilities,as do the expression products, antibodies elicited thereby, and DNA fromthese recombinants, as discussed above.

Example 22 Inocuity and the Immunogenicity of vCP205 in MacaquesInoculated by Intramuscular Route Experimental Animals:

Species: Macaca fascicularis (adult, wild caught)

Number: 8 Sex: Males

Origin: Mauritius Island considered free of Herpes B,Filovirus and Tuberculosis. Previous history: polio experiments.Diet and drinking water: Commercial diet and fruits; tap water adlibitum.

Four male Cynomolgus macaques were inoculated five times, at one monthintervals, with one dose of ALVAC-HIV (vCP205) (containing 10^(5.8)TCID₅₀/dose) by intramuscular route. The injections caused neithersymptoms nor lesions. Body weight of monkeys was not altered by theinjections. Four control animals were injected with placebo. (Mixture ofDMEM-F12 medium (25%), Lactoglutamate (25%) and freeze-drying substrate(50%)). General condition was monitored daily and injection sites wereobserved on days 1, 2, 3, 4 and 7 after each inoculation. Body weightswere recorded once a week.

Blood samples were collected in EDTA blood collection Vacutainer® tubes(Becton-Dickinson, Meyland, France) for hematological analyses, inlithium-heparin microvette CB 100 tubes (Sarstedt, Nümbrecht, Germany)for biochemical analysis and in tubes with serum separator SSTVacutainer® tubes (Becton-Dickinson, Meylan, France) for serologicalanalyses (MON:TSA.034.00). Samplings were done on days 0, 3, 7, 14, 28,31, 35, 42, 56, 59, 63, 70, 84, 87, 91, 98, 112, 115, 119, 126 and 140.Anesthesia (Ketamine, 20 mg/kg, intramuscular) was used for clinicalexaminations and blood sampling.

Hematological and biochemical analysis were carried out with the VETTEST 8008 Apparatus. Anti-HIV gp160, p24 proteins and V3 peptideantibodies were titered according to an ELISA technique. Maxisorp F96NUNC plates wells were coated for 1 hour at 37° C., with one of thefollowing antigens diluted in 0.1 M carbonate buffer, pH 9.6:

-   -   130 ng per well of purified gp160 MN/BRU (from recombinant        vaccinia containing 30% of cleaved gp160 and no alpha₂        macroglobulin),    -   200 ng of V3MN peptide,    -   130 ng of purified “p24”HIV (E. coli, P25 LAI isolate, batch        672Cl, Transgene).

All incubations were carried out in a final volume of 100 μl, followedby 3 washings performed with phosphate buffered saline, pH 7.1-0.1%Tween 20. Plates were blocked for 1 hour at 37° C. with 150 μl ofphosphate buffered saline pH 7.1-0.05% Tween 20-5% (W/V) powdered skimmilk (Gloria).

Serial threefold dilutions of the sera, ranging from 1/50 to 1/12150, inphosphate buffered saline—0.05% Tween 20-5% (W/V) powdered skim milk,were added to the wells and incubated for 90 min at 37° C.

After washing, anti-monkey IgG peroxidase conjugate (Cappel, goat IgGfraction), diluted 1/3000 in phosphate buffered saline—0.5% Tween 20-5%powdered skim milk, was added and the plates incubated for another 90min at 37° C. The plates, washed four times, were incubated in the darkfor 30 min at room temperature with the substrate O-phenylenediaminedihydrochloride (Sigma tablets). The substrate was used at theconcentration of 1.5 mg/ml in 0.05 M phosphate citrate buffer, pH 5.0containing 0.03 containing 0.03% sodium perborate (Sigma capsules).

The reactions were stopped with 50 μl of 4N H₂SO₄. The optical densitywas measured at 490-650 nm with an automatic plate reader (Vmax,Molecular Devices). The antibody titers were calculated according to theformula:

${Titer} = {\log \frac{{OD}_{490 - 650} \times 10}{1\text{/}{dilution}}\mspace{14mu} \left( {{OD}\mspace{14mu} {value}\mspace{14mu} {range}\text{:}\mspace{14mu} 0.2\mspace{14mu} {to}\mspace{14mu} 1} \right)}$

Neutralizing antibody assay determines the dilution of serum thatprevents the development of syncytia in 50% of microwells infected with10 TCID₅₀ of HIV MN. The MN strain was obtained from F. Barre-Sinoussiand propagated in CEM clone 166 cells.

Sera were decomplemented and twofold serial dilutions in RPMI beginning1/10 were prepared. Equal volumes of serum dilution and HIV suspension(500 ul each) were mixed and incubated for 2 h at 37° C. The HIVsuspension had been adjusted to contain 10² to 10^(2.5) TCID₅₀ per ml.

Prior to use, indicator CEMss cells were plated in microwells coatedwith poly-L-lysine, and incubated for 1 h at 37° C. Culture medium wasremoved and replaced with the virus/serum mixtures (100 μl/well, 6 wellsper dilution). After 1 h incubation at 37° C. culture medium was addedto each well and the plates were incubated at 37° C. All incubationswere in a CO₂ 5% incubator.

Seven and 14 days later, the cultures were examined under the microscopeand wells showing syncytia were recorded. Neutralizing 50% titer wascomputed according to SPEARMAN and KÄRBER and expressed as the log₁₀ ofthe end-point. As a confirmation, supernatants of the cultures werecollected on day seven, pooled for each dilution and assayed for RTactivity. Each assay includes an infectivity titration of the virussuspension, titration of antibody in a reference serum and a set ofuninfected microwells as negative controls.

Results:

A majority of the parameters selected to monitor hematological andbiochemical status varied within normal limits, including: erythrocytenumber, mean corpuscle volume, hematocrite, creatinin and alanineaminotransferase.

Some variations were seen, which cannot be attributed to the repeatedimmunizations with ALVAC-HIV (vCP205): i) unexplained lymphocytosis onweeks 8 and 9 in both the test and the control groups; ii) a decreasedlevel of hemoglobin in both groups on weeks 17 and 20 and in controlgroups; ii) a decreased level of hemoglobin in both groups on weeks 17and 20 and in controls on week 9; iii) erratic high values of theaspartate aminotransferase in two control monkeys and last; iv)irregular trombocytes counts caused by microcoagulation of somespecimens.

The anti-HIV immune response induced by vCP205 was assessed by ELISAtests using purified gp160 MN/BRU (from recombinant vaccinia), V3MNsynthetic peptide and p24 (from E. coli, LAI isolate). All the animalsdeveloped antibodies against gp160 and V3, and 2/4 against p24. Thepositive anti-HIV immune response usually appeared after 2, or maximum,3 injections. Subsequent vCP205 inoculations mainly maintained(sometimes slightly increased) the antibody levels and improvedhomogeneity of the response between macaques. Highest antibody titerswere usually observed two weeks after each inoculation, followed by adecrease until the next booster. Neutralizing antibodies against HIV/MNwere detectable in all immunized monkeys.

Clinical observation: neither erythema nor edema were reported at thesite of inoculation. Body weights were stable in control monkeys and inALVAC-HIV (vCP205) recipients (FIG. 32).

Hematological analyses: Leucocyte counts were greatly modified in bothcontrol and tested animals on weeks 8 and 9 with formula inversion (FIG.33 a). This fact, noted in both groups, is without correlation with theviral injections.

Erythrocyte number, corpuscle mean volume and hematocrite varied withinnormal limits but hemoglobin showed some discrepancy on weeks 9, 17 and20 in controls and on weeks 17 and 20 in animals inoculated withALVAC-HIV (vCP205) (FIGS. 33 b and c). Thrombocytes values varieddepending on the sampling quality (microcoagulation) (FIG. 33 c).

Biochemical analyses: Creatinin and ALAT (SGPTransaminase) did not varysignificantly after the reiterated inoculations FIGS. 34 and 36). TheAST (SGOTransaminase) values presented variations particularly importantin control monkeys #3 and 2 respectively on week 5 and 8-9 (FIG. 35).

Serological analyses (considering the ELISA titers of the negativecontrol group of macaques sera, the negative detection threshold of theserological response was considered to be, in log₁₀: 1.56±0.24,1.92±0.12 and 2.18±0.34, for gp160, V3 and p24 respectively): gp160 andV3 specific response (FIGS. 37 a-37 b, 38 a-38 b): the kinetics ofantibody of gp160 was similar to that to V3. The magnitude of the latterwas slightly weaker (mean titer at week 20 of 4.37 versus 8.84).

Two injections were necessary to induce a detectable immune response.Three monkeys showed a maximum response after the second injection; thefourth one required three (gp160) or four (V3) injections to do so.During the four week interval between injections, antibody titersconsistently increased then faded, to be boosted by the nextinoculation. While there were significant individual differences in theresponses to the initial injections, the responses leveled out as theexperiment progressed.

p24 a specific response (FIGS. 37 c, 38 c): a response was observed for2 macaques (macaques 5 and 7) out of 4 after respectively 2 and 3injections of vCP205. This is in contrast with a guinea-pig test inwhich no anti-p24 antibodies were detected in any inoculated animalgroups. As with anti-gp160 and anti-V3 antibodies, titers fluctuated upand down between injections and individual differences progressivelydisappeared. Unlike that of gp160 and V3, the anti-p24 antibody profiledid not reach a plateau.

Neutralizing antibody: as shown in FIG. 39, all four monkeys with vCP205developed detectable levels of neutralizing antibody against HIV (MN).One of them tested positive after one injection, two after the secondinjection and the last one required five administrations. It isnoteworthy that the latter animal also showed the slowest kinetics ofELISA antibody. The levels of neutralizing activity are relativelymodest (1/10 to 1/30 in arithmetical expression) and, likewise ELISAmeasurements, went up and down in the intervals between injections.

Effect of a late boost with the proteins gp160 and p24: The four monkeyswere boosted 7 weeks post the last injection of vCP205 with 200 μg ofgp160 and 200 μg of p24 proteins in incomplete Freund's adjuvant(Montanide ISA51) to raise hyperimmune reference sera; this caused apronounced increase (at least 10-fold) in antibody titers, suggestingthat the plateau seen in the ELISA analysis were not the limit ofresponse.

The immunization regimen induced high levels of binding antibody togp160 MN/BRU and V3 MN peptide, and low but definite neutralizingantibody. Serological results showed higher antibody levels than thoseobserved in macaques inoculated with ALVAC-HIV (vCP125) and one booster(instead of two) was sufficient to obtain a good anamnestic typeresponse. This Example shows that vCP205 and expression productsthereof, antibodies therefrom, and DNA from vCP205, can be used asdescribed above.

Example 23 Inocuity and Immunogenicity of vCP300 In MacaquesExperimental Animals:

Species: Macaca fascicularis

Number: 8 Sex: Males

Origin: Mauritius Island considered without Herpes B, Filovirus andtuberculosis.

Four male Cynomolgus macaques were immunized five times, at one monthintervals, with one dose of ALVAC-HIV (vCP300) (containing 10^(6.16)TCID₅₀/dose) by intramuscular route. Four control animals were injectedwith placebo. As a sixth injection, all the animals received ALVAC-HIV(vCP300) by intravenous route. The regimen was as follows:

Group #1 Product: Placebo

Route: Intramuscular alternately in the left or right deltoid muscle

Volume: 1 ml

Number of injections: 5 (on weeks 0, 4, 8, 12 and 16).

Group #2 Product: ALVAC-HIV

Route: Intramuscular alternately in the left or right deltoid muscle.

Volume: 1 ml. Dose: 10^(6.16) TCID₅₀.

Number of injections: 5 (on weeks 0, 4, 8, 12 and 16).

Groups #1 and #2

On week 20Product: ALVAC-HIV (vCP300)

Route: Intravenous Volume: 1 ml Dose: 106.16 TCID₅₀.

Number of injections: 1.

Clinical observations: Injection site was observed on days 1, 2, 3, 4and 7 following each inoculation. Animals were weighed once a week.Samplings: Blood samples were taken under ketamin anesthesia from thefemoral vein. Blood was collected in the following order in Vacutainertubes (Becton Dickinson, Meylan, France):

1) 1.8 ml in 0.129M buffered sodium citrated tube (prothrombine).2) 1 ml in 5 mg sodium fluoride and 4 mg potassium oxalate tube(glucose).3) 2 ml in 0.17M EDTA K₃ tube (hematological analyses).4) 2 to 3 ml in tubes for serum separation with inert barrier materialand clot activator (biochemical and serological analyses).

Samplings were done on days 0, 3, 7, 14, 28, 31, 35, 42, 56, 59, 63, 70,84, 87, 91, 98, 112, 119, 126, 140 and 143.

Dosages:

Hematological analyses included: blood counts and differential leucocytecounts, hemoglobin, thrombocytes, prothrombin, reticulocytes andsedimentation rate.

Biochemical analyses included: sodium, potassium, glucose, alkalinphosphatases, cholesterol, total proteins and electrophoresis,transaminases SGOT and SGPT.

Serological analyses: Anti-HIV gp160 glycoprotein, p24 protein, V3peptide and nef protein antibodies were titrated according to amodification of the ELISA technique:

Maxisorp F96 NUNC plates wells were coated for 1 hour at 37° C., thenovernight at 4° C., with one of the following antigens diluted in 0.1 Mcarbonate buffer, pH 9.6:

-   -   130 ng per well of purified gp160 MN/BRU (from recombinant        vaccinia VVTG 5156),    -   200 ng of V3MN peptide,    -   130 ng of purified p24 HIV (E. coli, p25 LAI isolate, batch        672Cl, Transgene).

All incubations were carried out in a final volume of 100 μl, followedby 3 washings performed with phosphate buffered saline, pH 7.1-0.1%Tween 20. Plates were blocked for 1 hour at 37° C. with 150 μl ofphosphate buffered saline pH 7.1-0.05% Tween 20-5% (W/V) powdered skimmilk (Gloria). Serial threefold dilutions of the sera, ranging from 1/50to 1/12150 or 1/500 to 1/121500, in phosphate buffered saline—0.05%Tween 20-5% (W/V) powdered skim milk, were added to the wells andincubated for 90 min. at 37° C.

After washing, anti-monkey IgG peroxidase conjugate (Cappel, goat IgGfraction), diluted 1/3000 in phosphate buffered saline—0.05% Tween 20-5%powdered skim milk, was added and the plates incubated for another 90min. at 37° C. The plates, washed four times, were incubated in the darkfor 30 min. at room temperature with the substrate O-phenylenediaminedihydrochloride (Sigma tablets), the substrate was used at theconcentration of 1.5 mg/ml in 0.05 M phosphate citrate buffer, pH 5.0containing 0.03% sodium perborate (Sigma capsules). The reactions werestopped with 50 μl of 4N H₂SO₄.

The optical density was measured at 490-650 nm with an automatic platereader (Vmax, Molecular Devices). The blanks were subtracted and thevalues of the duplicates averaged. The antibody titers were calculatedfor the OD value range of 0.2 to 1.2, from the regression curve of astandard anti-gp160 and anti-p24 hyperimmune serum of guinea-pig whichwas present on each ELISA plate. The titer of the standard serum hadbeen previously determined according to the formula:

${Titer} = {\log \frac{{OD}_{490 - 650} \times 10}{1\text{/}{dilution}}\mspace{14mu} \left( {{OD}\mspace{14mu} {value}\mspace{14mu} {range}\text{:}\mspace{14mu} 0.2\mspace{14mu} {to}\mspace{14mu} 1.2} \right)}$

Since no standard monkey anti-nef serum was available, the determinationof anti-nef antibodies was performed by including in the test areference monoclonal mouse antibody (anti-nef HIV1 ALI, MATG0020,Transgene) as an internal positive control. Anti-mouse IgG peroxidaseconjugate (Amersham) diluted 1/5000, was then used and sera titers werecalculated using the formula mentioned above.

Results:

The injections caused neither symptoms nor lesions. Body weight ofmonkeys was not altered by the injections. Hematological parameters didnot vary significantly and biochemical analyses showed no alteration ofkidneys and liver functions.

Hematological results: Variations, when present, were similar in patternand in range in the placebo and in the vaccinee groups.

Individual WBC counts varied within normal limits. Differential WBCcounts often showed a diminution of lyphocytes 3 days after the bloodsamplings (FIG. 40). Erythrocyte counts, hematocrite and hemoglobintransiently decreased after each blood sampling contrary to reticulocytecounts which increased regularly after the puncture (FIGS. 41 a, 41 b,41 c). Mean corpuscle volume was stable in all the animals except anincrease on day 140. Thrombocyte counts showed some variations (FIG. 41)but prothrombin level was stable (FIG. 42). There was no sign of anemiain either group. Sedimentation rate was 1 mm after one hour in all thesamples.

Biochemical results: For a better interpretation of the variationsobserved, a standard serum (pool of 18 macaque sera) was analyzed at thebeginning (Std a) and at the end (Std p) of each series of samples to betested.

Cholesterol values varied in the same way in controls and in test group(FIGS. 43 a and 43 b).

Sodium and potassium were stable after all the injections but, as fortotal proteins and glucose, there was a great rising on day 140. On thatday, blood specimens were drawn prior to the intravenous injectionwithout anesthesia (so that clinical reactions could be monitoredwithout interference); the change observed in several biologicalparameters was likely due to the stress associated with handling andsampling in the absence of anesthesia (FIGS. 43 a and 43 b).Electrophoresis profiles (data not shown) were very similar in all thesamples. Creatinine, bilirubin, glutamic oxaloacetic and glutamicpyruvic transaminases and alkalin phosphatases varied within normallimits and always in the same direction in both control and test groups(FIGS. 43 c and 43 d). Kidneys and liver functions were therefore notaffected by the inoculations of ALVAV-HIV (vCP300).

Serological results: Two (macaques # 6, 7, 8) or three (macaque #5)injections were necessary to induce detectable anti-gp160 and anti-V3responses (FIG. 44 a, 44 b, 45 a, 45 b). Two weeks after the secondinjection, these responses were variable between animals (anti-gp160titers fluctuating between 2 to 4.6 logs). This response heterogeneitywas smoothed out by the third injection. The subsequent intramuscularinjections mainly maintained or increased the titers which reachedaround 4.3 and 3.6 for respectively anti-gp160 and anti-V3 antibodiesafter the fourth to fifth injection. Detectable anti-p24 antibodies(FIGS. 46 a, 46 b) were observed after three (macaques # 6, 8) to four(macaque #7) or five (macaque 5) vCP300 intramuscular injections. Theanimals with the highest anti-gp160/V3 titers exhibited the highestanti-p24 (≈4.3 on week 18 post-primoimmunization). None of the animalsraised anti-nef antibodies (FIG. 47 a, 47 b).

The immune response induced by vCP300 was assessed by analyzing in ELISAthe anti-HIV-1 gp160, V3, p24 and nef sera antibodies.

All the animals developed antibodies against gp160, V3 and p24:significant anti-gp160 or V3 responses were obtained after 2 or at most3 intramuscular injections. Subsequent inoculations maintained orincreased the antibody levels. Anti-p24 responses were detected after 3to 5 injections and each inoculation of vCP300 increased the levels. Noanti-nef antibodies could be detected in any of the animals.

The highest antibody titers were usually observed two weeks after eachintramuscular injection, followed by a decrease until the next boost.

These serological results are very close to those obtained withALVAC-HIV (vCP205) which expressed the same proteins but no CTLepitopes. Two minor differences can be pointed out in this example withvCP300: slightly lower anti-gp160/V3 antibody titers (≈0.2 to 0.5 log),and higher anti-p24 responses (all macaques positive, higher seratiters). Because of the individual variations between animals, thesedifferences were not deemed significant. No indication ofhypersensitivity was seen following intravenous inoculation. No sideeffects were recorded. This regimen induced high levels of bindingantibodies to gp160 (though lower than with vCP205), V3 and p24antigens. This Example shows that vCP300 and expression productsthereof, antibodies therefrom, and DNA from vCP300 can be used asdescribed above.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited by particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope thereof.

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1-35. (canceled)
 36. A protein having an amino acid sequence comprisingthe amino acid sequence of HIV1 gp120, wherein the amino acid sequenceof the gp120 is modified so as to contain an epitope not naturallyoccurring in HIV1 gp120.
 37. The protein of claim 36, wherein theepitope is a B-cell epitope.
 38. The protein of claim 36, wherein theamino acid sequence of the gp120 is modified in the V3 loop so as tocontain the epitope.
 39. The protein of claim 38, wherein the epitope isa B-cell epitope.
 40. The protein of claim 38, wherein the epitope isELDKWA or LDKW.
 41. The protein of claim 36, wherein the amino acidsequence of the gp120 is modified to contain at least one of HIV1gag(+pro) (IIIB), gp120(MN) (+ transmembrane), nef(BRU)CTL, pol(IIIB)CTL, andELDKWA or LDKW epitopes.
 42. The protein of claim 41, wherein the aminoacid sequence of the gp120 is modified in the V3 loop to contain theepitope.
 43. The protein of claim 38, comprising the amino acid sequenceidentified as SEQ ID NO: 137, SEQ ID NO: 140 or SEQ ID NO:
 143. 44. Theprotein having the amino acid sequence identified as SEQ ID NO: 137, SEQID NO: 140 or SEQ ID NO:
 143. 45. A nucleic acid molecule comprising anucleic acid sequence which encodes the protein of claim
 36. 46. Anucleic acid molecule comprising a nucleic acid sequence which encodesthe protein of claim
 37. 47. A nucleic acid molecule comprising anucleic acid sequence which encodes the protein of claim
 38. 48. Anucleic acid molecule comprising a nucleic acid sequence which encodesthe protein of claim
 39. 49. A nucleic acid molecule comprising anucleic acid sequence which encodes the protein of claim
 40. 50. Anucleic acid molecule comprising a nucleic acid sequence which encodesthe protein of claim
 41. 51. A nucleic acid molecule comprising anucleic acid sequence which encodes the protein of claim
 42. 52. Anucleic acid molecule comprising the nucleic acid sequence which encodesthe amino acid sequence identified as SEQ ID NO: 137, SEQ ID NO: 140 orSEQ ID NO:
 143. 53. The nucleic acid molecule of claim 52 selected fromthe group consisting of the nucleic acid molecules identified as SEQ IDNO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 141and SEQ ID NO: 142.